Assay panels

ABSTRACT

Described herein are kits and components thereof used for a multiplexed analysis of a set of cytokines.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of U.S. Provisional Application No. 61/748,626 filed on Jan. 3, 2013, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to kits used for the detection of cytokines using electrochemiluminescent technology.

BACKGROUND OF THE INVENTION

Cytokines are the soluble factors that mediate acute and chronic inflammatory responses, and are involved in many physiological events from wound healing to autoimmune disorders. They are important regulators of cell-mediated and humoral immune responses and their differential expression has been associated with a wide array of immune disorders. They function on a variety of cell types, having stimulatory or inhibitory effects on proliferation, differentiation, and maturation. Therefore, measuring the level of only a single cytokine in any biological system provides only partial information relevant to the response on a systemic level. Comprehensive tests for cytokine levels generally aim to measure the concentrations of a large set of cytokines to gain a better understanding of the underlying physiology.

The enzyme-linked immunosorbent assay (ELISA) is the most commonly used and reported method for the quantitation of secreted cytokines. However, ELISA can only detect one analyte per reaction in individual assay wells. This leads to high reagent cost, excessive technician time, and the need to use large sample volumes to generate each results. The ability to detect and quantitate many cytokines simultaneously in the same sample via a robust multiplexed assay would reduce costs and improve efficiency. The advantages of multiplex technology over conventional assay methods include simultaneous analyte detection, reduced reagent handling, high assay throughput, and decreased sample and reagent volumes.

SUMMARY OF THE INVENTION

The invention provides a kit for the analysis of a cytokine panel comprising:

i. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFalpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

ii. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

iii. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, MCP-4; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins;

iv. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following rat analytes are bound: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins; or

v. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following mouse analytes are bound: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

Also provided is a method of manufacturing a kit or a lot of kit such as those described herein that includes: (a) subjecting a preliminary set of detection antibodies specific for said human analytes to CIEF, DLS, and Experion; (b) selecting qualified detection antibodies from said preliminary set of detection antibodies based on said CIEF, DLS, and Experion testing; (c) subjecting a preliminary set of capture antibodies specific for said human analytes to CIEF, DLS, and Experion; and (b) selecting qualified capture antibodies from said preliminary set of capture antibodies based on said CIEF, DLS, and Experion testing. In a preferred embodiment, a lot of kits is manufacturing using this protocol and meets one or more of the following specifications: (a) average intraplate CV of ≦10%; (b) maximum intraplate CV of ≦13%; (c) average uniformity metric of ≦25%; (d) maximum uniformity metric of ≦37%; (e) CV of intraplate averages of ≦18%; (f) lower signal boundary of >1500; and (g) upper signal boundary of <10⁶.

In a preferred embodiment, the invention provides a kit for the analysis of two or more cytokine panels comprising: (a) two or more multi-well assay plates each comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to a set of analytes are bound, wherein said set of analytes is selected from the group consisting of:

(i) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha;

(ii) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A;

(iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4;

(iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or

(v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha;

(b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

An additional embodiment of the invention is a 10-spot 96-well multi-well plate, wherein each plate comprises a plate top, a plate bottom, an x- and y-axis of the plate top and bottom, and each well comprises a spot pattern, wherein the plate meets the following specifications: Δx≦0.2 mm, Δy≦0.2 mm, and α≦0.1°, wherein (a) Δx is the difference between a center of the spot pattern and a center of a well along the x axis of the plate; (b) Δy is the difference between the center of a spot pattern and a center of the well along the y axis of the plate; and (c) α is a counter-clockwise angle between the x axis of the plate bottom and the x axis of the plate top.

Moreover, the invention contemplates a 10-spot 96-well multi-well plate, wherein each plate comprises a plate top, a plate bottom, and each well comprises a spot pattern, wherein the plate meets the following specifications: (a) a length range of 3.8904-3.9004 inches; (b) a width range of 2.4736-2.4836 inches; and (c) well to well spacing of 0.3513-0.3573 inches.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1( a)-(c) illustrate a 10-spot pattern in a well of a multi-well plate (panel (a)), its placement in a 96-well 10-spot plate (panel (b)), and the principles of an immunoassay conducted using a multi-well assay plate such as those described herein.

FIGS. 2( a)-(e) are standard curves for each of the five cytokine panels.

FIGS. 3( a)-(b) shows the configuration of a 96 well multi-well assay plate.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

As used herein, the term “sample” is intended to mean any biological fluid, cell, tissue, organ or combinations or portions thereof, which includes or potentially includes a biomarker of a disease of interest. For example, a sample can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture. A sample further can be a subcellular fraction or extract, or a crude or substantially pure nucleic acid molecule or protein preparation. In one embodiment, the samples that are analyzed in the assays of the present invention are blood, peripheral blood mononuclear cells (PBMC), isolated blood cells, serum and plasma. Other suitable samples include biopsy tissue, intestinal mucosa, saliva, cerebral spinal fluid, and urine.

The present invention relates to a kit for the analysis of a cytokine panel. At least five assay panels are contemplated and each kit is configured to analyze one of the following panels:

TABLE 1 Cytokine Assay Panels Panel Species Analytes 1 Human IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-13, TNFalpha 2 Human GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A 3 Human Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8 , MCP-1, MDC, MCP-4 4 Rat IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha 5 Mouse IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha

The kits can include (a) a single panel arrayed on a multi-well plate which is configured to be used in an electrochemiluminescence assay, as well as (b) associated consumables, e.g., detection antibodies, calibrators, and optional diluents and/or buffers. Alternatively, the multi-well plates and associated consumables can be provided separately. Still further, a kit can include two or more multi-well plates with panels arrayed thereon, i.e., panels 1-5, and the associated consumables can be provided in the kit or separately.

Panels 1, 2, 4, and 5 include inflammation-related and/or growth factor biomarkers that are important for inflammation response, immunity, and regulation of numerous biological processes. These secreted biomarkers can be detected in a variety of tissues and bodily fluids and their over- or under-expression can indicate a shift in biological equilibrium of the body. These panels also consist of many of the Th1/Th2 pathway biomarkers. The biomarkers in these panels are involved in numerous disorders such as rheumatoid arthritis, Alzheimer's disease, asthma, atherosclerosis, allergies, systematic lupus erythematosus, obesity, cancer, depression, multiple sclerosis, diabetes, psoriasis, and Crohn's disease, among others.

Panel 3 consists of eight CC chemokine assays (MCP-1, MIP-1a, MIP-1b, Eotaxin, MCP-4, TARC, MDC, and Eotaxin-3) and two CXC chemokine assays (IL-8 and IP-10). Chemokines are small chemotactic cytokines with molecular weights around 8-10 kDa that are capable of inducing directed chemotaxis. The four cysteine residues in conserved locations result in their compact 3-dimensional structure. Based on the spacing of the first two cysteine residues, they are divided into four families of chemokines—CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines, where C represents cysteine and X represents any other amino acids. Chemokines function by activating specific G protein-coupled receptors resulting in migration of inflammatory and non-inflammatory cells. The pro-inflammatory chemokines are responsible for migration of immune cells to the infection site while the homeostatic chemokines are responsible for the migration of cells for the purpose of tissue maintenance and development. Chemokines are associated with number of diseases.

Panels 1-5 are configured in a multi-well assay plate including a plurality of wells, each well having an array with 10 “spots” or discrete binding domains. An example of a 10-spot well is shown in FIG. 1( a) and the incorporation of that well into a multi-well plate is shown in FIG. 1( b). A capture antibody to each analyte is immobilized on a binding domain in the well and that capture antibody is used to detect the presence of the target analyte in an immunoassay as illustrated in FIG. 1( c). Briefly, a sample suspected of containing that analyte is added to the well and if present, the analyte binds to the capture antibody at the designated binding domain. The presence bound analyte on the binding domain is detected by adding labeled detection antibody. The detection antibody also binds to the analyte forming a “sandwich” complex (capture antibody—analyte—detection antibody) on the binding domain. The location of each analyte in Panels 1-5 in this 10-spot pattern is identified in Table 2.

TABLE 2 Spot Pattern Configuration Per Panel Panel Species Spot Location Analytes 1 Human 1 IFN-gamma 2 IL-1beta 3 IL-2 4 IL-4 5 IL-6 6 IL-8 7 IL-10 8 IL-12p70 9 IL-13 10 TNFalpha 2 Human 1 GM-CSF 2 IL-1alpha 3 IL-5 4 IL-7 5 IL-12/IL-23 p40 6 IL-15 7 IL-16 8 IL-17A 9 TNF-beta 10 VEGF-A 3 Human 1 Eotaxin, 2 MIP-1 alpha, 3 Eotaxin-3 4 TARC 5 IP-10 6 MIP-1 beta 7 IL-8 8 MCP-1 9 MDC 10 MCP-4 4 Rat 1 IFN-gamma 2 IL-2 3 IL-4 4 IL-1 beta 5 IL-5 6 IL-6 7 KC/GRO 8 IL-10 9 IL-13 10 TNF-alpha 5 Mouse 1 IFN-gamma 2 IL-1-beta 3 IL-2 4 IL-4 5 IL-5 6 IL-6 7 KC/GRO 8 IL-10 9 IL-12p70 10 TNF-alpha

The multiplexed immunoassay kits described herein allow a user to simultaneously quantify multiple biomarkers. The panels are selected and optimized such that the individual assays function well together. The sample may require dilution prior to being assayed. Sample dilutions for specific sample matrices of interest are optimized for a given panel to minimize sample matrix effects and to maximize the likelihood that all the analytes in the panel will be within the dynamic range of the assay. In a preferred embodiment, all of the analytes in the panel are analyzed with the same sample dilution in at least one sample type. In another preferred embodiment, all of the analytes in a panel are measured using the same dilution for most sample types.

For a given panel, the detection antibody concentration and the number of labels per protein (L/P ratio) for the detection antibody are adjusted to bring the expected levels of all analytes into a quantifiable range at the same sample dilution. If one wants to increase the high end of the quantifiable range for a given analyte, then the L/P can be decreased and/or the detection antibody concentration is decreased. On the other hand, if one wants to increase the lower end of the quantifiable range, the L/P can be increased, the detection antibody concentration can be increased if it is not at the saturation level, and/or the background signal can be lowered.

Calibration standards for use with the assay panels are selected to provide the appropriate quantifiable range with the recommended sample dilution for the panel. The calibration standards have known concentrations of one of more of the analytes in the panel. Concentrations of the analytes in unknown samples are determined by comparison to these standards. In one embodiment, calibration standards comprise mixtures of the different analytes measured by an assay panel. Preferably, the analyte levels in a combined calibrator are selected such that the assay signals for each analyte are comparable, e.g., within a factor of two, a factor of five or a factor of 10. In another embodiment, calibration standards include mixtures of analytes from multiple different assay panels.

A calibration curve may be fit to the assay signals measured with calibration standards using, e.g., curve fits known in the art such as linear fits, 4-parameter logistic (4-PL) and 5-parameter (5-PL) fits. Using such fits, the concentration of analytes in an unknown sample may be determined by backfitting the measured assay signals to the calculated fits. Measurements with calibration standards may also be used to determine assay characteristics such as the limit of detection (LOD), limit of quantification (LOQ), dynamic range, and limit of linearity (LOL).

A kit includes the following assay components: a multi-well assay plate configured to conduct an immunoassay for one of the panels described herein, a set of detection antibodies for the analytes in the panel (wherein the set comprises individual detection antibodies and/or a composition comprising a blend of one or more individual detection antibodies), and a set of calibrators for the analytes in the panel (wherein the set comprises individual calibrator protein compositions and/or a composition comprising a blend of one or more individual calibrator proteins). The kit can also include one of more of the following additional components: a blocking buffer (used to block assay plates prior to addition of sample), an antibody diluent (used to dilute stock detection antibody concentrations to the working concentration), an assay diluent (used to dilute samples), a calibrator diluent (used to dilute or reconstitute calibration standards) and a read buffer (used to provide the appropriate environment for detection of assay labels, e.g., by an ECL measurement). The antibody and assay diluents are selected to reduce background, optimize specific signal, and reduce assay interference and matrix effect. The calibrator diluent is optimized to yield the longest shelf life and retention of calibrator activity. The blocking buffer should be optimized to reduce background. The read buffer is selected to yield the appropriate sensitivity, quantifiable range, and slowest off-rate. The reagent components of the kit can be provided as liquid reagents, lyophilized, or combinations thereof, diluted or undiluted, and the kit includes instructions for appropriate preparation of reagents prior to use. In a preferred embodiment, a set of detection antibodies are included in the kit comprising a plurality of individual detection antibody compositions in liquid form. Moreover, the set of calibrators provided in the kit preferably comprise a lyophilized blend of calibrator proteins. Still further, the kit includes a multi-well assay plate that has been pre-coated with capture antibodies and exposed to a stabilizing treatment to ensure the integrity and stability of the immobilized antibodies.

As part of a multiplexed panel development, assays are optimized to reduce calibrator and detection antibody non-specific binding. In sandwich immunoassays, specificity mainly comes from capture antibody binding. Some considerations for evaluating multiplexed panels include: (a) detection antibody non-specific binding to capture antibodies is reduced to lower background of assays in the panel, and this can be achieved by adjusting the concentrations and L/P of the detection antibodies; (b) non-specific binding of detection antibodies to other calibrators in the panel is also undesirable and should be minimized; (c) non-specific binding of other calibrators in the panel and other related analytes should be minimized; if there is calibrator non-specific binding, it can reduce the overall specificity of the assays in the panel and it can also yield unreliable results as there will be calibrator competition to bind the capture antibody.

Different assays in the panel may require different incubation times and sample handling requirements for optimal performance. Therefore, the goal is to select a protocol that's optimized for most assays in the panel. Optimization of the assay protocol includes, but is not limited to, adjusting one or more of the following protocol parameters: timing (incubation time of each step), preparation procedure (calibrators, samples, controls, etc.), and number of wash steps.

The reagents used in the kits, e.g., the detection and capture antibodies and calibrator proteins, are preferably subjected to analytical testing and meet or exceed the specifications for those tests. The analytical tests that can be used to characterize kit materials include but are not limited to, CIEF, DLS, reducing and/or non-reducing EXPERION, denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof. In a preferred embodiment, the materials are characterized by CIEF, DLS, and reducing and non-reducing EXPERION. One or more additional tests, including but not limited to denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof, can also be used to characterize the materials. In a preferred embodiment, the materials are also subjected to functional testing, i.e., a binding assay for the target analyte, as well as one or more characterization tests, such as those listed above. If the materials do not meet or exceed the specifications for the functional and/or characterization tests, they can be subjected to additional purification steps and re-tested. Each of these tests and the metrics applied to the analysis of raw materials subjected to these tests are described below:

Capillary Isoelectric Focusing (CIEF) is a technique commonly used to separate peptides and proteins, and it is useful in the detection of aggregates. During a CIEF separation, a capillary is filled with the sample in solution and when voltage is applied, the ions migrate to a region where they become neutral (pH=pI). The anodic end of the capillary sits in acidic solution (low pH), while the cathodic end sits in basic solution (high pH). Compounds of equal isoelectric points (pI) are “focused” into sharp segments and remain in their specific zone, which allows for their distinct detection based on molecular charge and isoelectric point. Each specific antibody solution will have a fingerprint CIEF that can change over time. When a protein solution deteriorates, the nature of the protein and the charge distribution can change. Therefore, CIEF is a particularly useful tool to assess the relative purity of a protein solution and it is a preferred method of characterizing the antibodies and calibrators in the plates and kits described herein. The metrics used in CIEF include pI of the main peak, the pI range of the solution, and the profile shape, and each of these measurements are compared to that of a reference standard.

Dynamic Light Scattering (DLS) is used to probe the diffusion of particulate materials either in solution or in suspension. By determining the rate of diffusion (the diffusion coefficient), information regarding the size of particles, the conformation of macromolecular chains, various interactions among the constituents in the solution or suspension, and even the kinetics of the scatterers can be obtained without the need for calibration. In a DLS experiment, the fluctuations (temporal variation, typically in a μs to ms time scale) of the scattered light from scatterers in a medium are recorded and analyzed in correlation delay time domain. Like CIEF, each protein solution will generate a fingerprint DLS for the particle size and it's ideally suited to detect aggregation. All IgGs, regardless of binding specificity, will exhibit the same DLS particle size. The metrics used to analyze a protein solution using DLS include percentage polydispersity, percentage intensity, percentage mass, and the radius of the protein peak. In a preferred embodiment, an antibody solution meets or exceeds one or more of the following DLS specifications: (a) radius of the antibody peak: 4-8 nm (antibody molecule size); (b) polydispersity of the antibody peak: <40% (measure of size heterogeneity of antibody molecules); (c) intensity of the antibody peak: >50% (if other peaks are present, then the antibody peak is the predominant peak); and (d) mass in the antibody peak: >50%.

Reducing and non-reducing gel electrophoresis are techniques well known in the art. The EXPERION™ (Bio-Rad Laboratories, Inc., www.bio-rad.com) automated electrophoresis station performs all of the steps of gel-based electrophoresis in one unit by automating and combining electrophoresis, staining, destaining, band detection, and imaging into a single step. It can be used to measure purity. Preferably, an antibody preparation is greater 50% pure by Experion, more preferably, greater than 75% pure, and most preferably greater than 80% pure. Metrics that are applied to protein analysis using non-reducing Experion include percentage total mass of protein, and for reducing Experion they include percentage total mass of the heavy and light chains in an antibody solution, and the heavy to light chain ratio.

Multi-Angle Light Scattering (MALS) detection can be used in the stand-alone (batch) mode to measure specific or non-specific protein interactions, as well as in conjunction with a separation system such as flow field flow fractionation (FFF) or size exclusion chromatography (SEC). The combined SEC-MALS method has many applications, such as the confirmation of the oligomeric state of a protein, quantification of protein aggregation, and determination of protein conjugate stoichiometry. Preferably, this method is used to detect molecular weight of the components of a sample.

In a preferred embodiment, an assay is conducted in a single assay chamber, such as a single well of an assay plate or an assay chamber that is an assay chamber of a cartridge. In a preferred embodiment, the kits of the invention include multi-well assay plates that are configured to conduct an electrochemiluminescence measurement as described for example, in US 20040022677; US 20050052646; US 20050142033; US 20040189311, each of which is incorporated herein by reference in their entireties. Assay plates and plate readers are now commercially available (MULTI-SPOT® and MULTI-ARRAY® plates and SECTOR® instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg, Md.).

As used herein, a lot of kits comprise a group of kits comprising kit components that meet a set of kit release specifications. A lot can include at least 10, at least 100, at least 500, at least 1,000, at least 5,000, or at least 10,000 kits and a subset of kits from that lot are subjected to analytical testing to ensure that the lot meets or exceeds the release specifications. In one embodiment, the release specifications include but are not limited to kit processing, reagent stability, and kit component storage condition specifications. Kit processing specifications include the maximum total sample incubation time and the maximum total time to complete an assay using the kit. Reagent stability specifications include the minimum stability of each reagent component of the kit at a specified storage temperature. Kit storage condition specifications include the range of storage temperatures for all components of the kit, the maximum storage temperature for frozen components of the kit, and the maximum storage temperature for non-frozen components of the kit. A subset of kits in a lot are reviewed in relation to these specifications and the size of the subset depends on the lot size. In a preferred embodiment, for a lot of up to 300 kits, a sampling of 4-7 kits are tested; for a lot of 300-950 kits, a sampling of 8-10 kits are tested; and for a lot of greater than 950 kits, a sampling of 10-12 kits are tested. Alternatively or additionally, a sampling of up to 1-5% preferably up to 1-3%, and most preferably up to 2% is tested.

In addition, each lot of multi-well assay plates is preferably subjected to uniformity and functional testing. A subset of plates in a lot are subjected to these testing methods and the size of the subset depends on the lot size. In a preferred embodiment, for a lot of up to 300 plates, a sampling of 4-7 plates are tested; for a lot of 300-950 plates, a sampling of 8-10 plates are tested; and for a lot of greater than 950 plates, a sampling of 10-12 plates are tested. Alternatively or additionally, a sampling of up to 1-5% preferably up to 1-3%, and most preferably up to 2% is tested. The uniformity and functional testing specifications are expressed in terms of % CV, Coefficient of Variability, which is a dimensionless number defined as the standard deviation of a set of measurements, in this case, the relative signal detected from binding domains across a plate, divided by the mean of the set.

One type of uniformity testing is protein A/G testing. Protein A/G binding is used to confirm that all binding domains within a plate are coupled to capture antibody. Protein A/G is a recombinant fusion protein that combines IgG binding domains of Protein A and protein G and it binds to all subclasses of human IgG, as well as IgA, IgE, IgM and, to a lesser extent, IgD. Protein A/G also binds to all subclasses of mouse IgG but not mouse IgA, IgM, or serum albumin, making it particularly well suited to detect mouse monoclonal IgG antibodies without interference from IgA, IgM, and serum albumin that might be present in the sample matrix. Protein A/G can be labeled with a detectable moiety, e.g., a fluorescent, chemiluminescent, or electrochemiluminescent label, preferably an ECL label, to facilitate detection. Therefore, if capture antibody is adhered to a binding domain of a well, it will bind to labeled protein NG, and the relative amount of capture antibody bound to the surface across a plate can be measured.

In addition to the uniformity testing described above, a uniformity metric for a subset of plates within a lot can be calculated to assess within-plate trending. A uniformity metric is calculated using a matrix of normalized signals from protein NG and/or other uniformity or functional tests. The raw signal data is smoothed by techniques known in the art, thereby subtracting noise from the raw data, and the uniformity metric is calculated by subtracting the minimum signal in the adjusted data set from the maximum signal.

In a preferred embodiment, a subset of plates in a lot is subjected to protein A/G and functional testing and that subset meet or exceed the following specifications:

TABLE 3(a) Plate Metrics Preferred Specification for a Metric subset of 96 well multi-well plates Average intraplate CV ≦10% Maximum intraplate CV ≦13% Average Uniformity ≦25% Maximum Uniformity ≦37% CV of intraplate averages ≦18% Signal, lower boundary >1500 Signal, upper boundary <10⁽⁶⁾

As disclosed in U.S. Pat. No. 7,842,246 to Wohlstadter et al., the disclosure of which is incorporated herein by reference in its entirety, each plate consists of several elements, e.g., a plate top, a plate bottom, wells, working electrodes, counter electrodes, reference electrodes, dielectric materials, electrical connects, and assay reagents. The wells of the plate are defined by holes/openings in the plate top. The plate bottom can be affixed, manually or by automated means, to the plate top, and the plate bottom can serve as the bottom of the well. Plates may have any number of wells of any size or shape, arranged in any pattern or configuration, and they can be composed of a variety of different materials. Preferred embodiments of the invention use industry standard formats for the number, size, shape, and configuration of the plate and wells. Examples of standard formats include 96, 384, 1536, and 9600 well plates, with the wells configured in two-dimensional arrays. Other formats may include single well plates (preferably having a plurality of assay domains that form spot patterns within each well), 2 well plates, 6 well plates, 24 well plates, and 6144 well plates. Each well of the plate includes a spot pattern of varying density, ranging from one spot within a well to 2, 4, 7, 9, 10, 16, 25, etc. In a preferred embodiment, the plates used in the kits of the invention comprise 10-spot 96-well plates.

Each plate is assembled according to a set of preferred specifications. In a preferred embodiment, a plate bottom meets or exceeds the following specifications:

TABLE 3(b) Plate bottom specifications 96-well (round well) specifications in Parameter inches Length range (C to C)* 3.8904-3.9004 (A1-A12 and H1-H12)** Width range (C to C) 2.4736-2.4836 (A1-A12 and H1-H12) Well to well spacing 0.3513-0.3573 *C to C well distance is the center of spot to center of spot distance between the outermost wells of a plate. **As shown in FIG. 3, a 96-well multi-well plate includes a set of wells arranged in an 8 × 12 array, wherein the rows on the short side of the plate are identified by A-H, and the columns on the long side of the plate are identified by 1-12. Therefore, length and width can be measured in row A1-A12 and compared to that of row H1-H12.

In a further preferred embodiment, the plate also meets or exceeds defined specifications for alignment of a spot pattern within a well of the plate. These specifications include three parameters: (a) Δx, the difference between the center of the spot pattern and the center of the well along the x axis of the plate (column-wise, long axis); (b) Δy, the difference between the center of the spot pattern and the center of the well along the y axis of the plate (row-wise, short axis); and (c) α, the counter-clockwise angle between the long axis of the plate bottom and the long axis of the plate top of a 96-well plate. In a preferred embodiment, the plate meets or exceeds the following specifications: Δx≦0.2 mm, Δy≦0.2 mm, and α≦0.1°.

The following non-limiting examples serve to illustrate rather than limit the present invention.

Examples Example 1 Reagent Preparation

All reagents were brought to room temperature and diluents were thawed in water at room temperature.

(i) Preparation of Standards

Multi-analyte lyophilized calibrator blends and all diluents for each panel were obtained from Meso Scale Discovery (Rockville, Md.) which yield the recommended highest standard upon reconstitution in one mL of diluent. The lyophilized calibrator was reconstituted and kept on ice. Seven (7) standard solutions and a zero calibrator blank were prepared for up to 4 replicates as follows: (x) The highest standard was prepared by adding 1000 μL of diluent to the lyophilized calibrator vial. The solution was mixed by vortexing and keep on wet ice for a minimum of 5 minutes prior to use. (y) The next standard was prepared by transferring 75 μL of the highest standard to 225 μL of diluent. The solution was mixed well and the procedure repeated 4-fold serial dilutions 5 additional times to generate 7 standards. (z) Diluent was used as the blank. Once reconstituted to the recommended highest standard in Diluent 2, the multi-analyte lyophilized calibrator for each kit is stable at 2-8° C. for 30 days.

(ii) Sample Collection & Handling

When preparing serum, samples were allowed to clot for two hours at room temperature. Plasma prepared in heparin tubes commonly display additional clotting following thawing of the sample. Both serum and plasma were centrifuged for 20 minutes at 2000×g prior to aliquoting. For serum-free medium, the presence of carrier proteins, e.g., 1% BSA, in the solution was used to prevent loss of analyte to the labware. Samples with extremely high levels of cytokines were diluted. Tissue culture supernatant samples were diluted at least 2-fold in diluent. Upon collection, samples were tested immediately or aliquots were frozen at ≦20° C. Samples were centrifuged at 2000 g for three minutes to remove particulates prior to sample preparation.

(iii) Dilution of Samples

For human serum, plasma, CSF, urine, and cell culture supernates, a minimum of 2-fold dilution in diluent was done.

(iv) Preparation of Controls

Controls were prepared in non-human animal matrix with spiked recombinant human analytes. The lyophilized controls were reconstituted in 250 uL of diluent and treated as a sample. Once reconstituted in 250 uL of diluent, the controls were stable for 30 days at 2-8° C.

(v) Preparation of Detection Antibody Solutions

Detection antibodies were obtained from Meso Scale Discovery (Rockville, Md.) as a 50× stock solution and the working detection antibody solution was 1×. Exposure of 1× detection antibody solution to light was avoided to prevent elevated assay background. Once prepared, the 1× detection antibody solution was kept in the dark.

For 1 plate of Panel 1, the following were combined:

-   -   1. 60 uL of 50× SULFO-TAG™ Anti-human IFN-gamma antibody     -   2. 60 uL of 50× SULFO-TAG Anti-human IL-1beta antibody     -   3. 60 uL of 50× SULFO-TAG Anti-human IL-2 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-human IL-4 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-human IL-6 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-human IL-8 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-human IL-10 antibody     -   8. 60 uL of 50× SULFO-TAG Anti-human IL-12p70 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-human IL-13 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-human TNFalpha antibody     -   11. 2400 uL Diluent 3 from Meso Scale Discovery (Rockville, Md.)         For 1 plate of Panel 2, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-human GM-CSF antibody     -   2. 60 uL of 50× SULFO-TAG Anti-human IL-1 alpha antibody     -   3. 60 uL of 50× SULFO-TAG Anti-human IL-5 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-human IL-7 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-human IL-12/IL-23p40 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-human IL-15 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-human IL-16 antibody     -   8. 60 uL of 50× SULFO-TAG Anti-human IL-17A antibody     -   9. 60 uL of 50× SULFO-TAG Anti-human TNFbeta antibody     -   10. 60 uL of 50× SULFO-TAG Anti-human VEGF-A antibody     -   11. 2400 uL Diluent 3 from Meso Scale Discovery (Rockville, Md.)         For 1 plate of Panel 3, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-human Eotaxin antibody     -   2. 60 uL of 50× SULFO-TAG Anti-human MIP-1beta antibody     -   3. 60 uL of 50× SULFO-TAG Anti-human MCP-4 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-human Eotaxin-3 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-human TARC antibody     -   6. 60 uL of 50× SULFO-TAG Anti-human IP-10 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-human MIP-1alpha antibody     -   8. 60 uL of 50× SULFO-TAG Anti-human IL-8 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-human MCP-1 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-human MDC antibody     -   11. 2400 uL Diluent 3 from Meso Scale Discovery (Rockville, Md.)         For 1 plate of Panel 4, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-rat IFN-gamma antibody     -   2. 60 uL of 50× SULFO-TAG Anti-rat IL-2 antibody     -   3. 60 uL of 50× SULFO-TAG Anti-rat IL-4 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-rat IL-1 beta antibody     -   5. 60 uL of 50× SULFO-TAG Anti-rat IL-5 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-rat IP-6 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-rat KC/GRO antibody     -   8. 60 uL of 50× SULFO-TAG Anti-rat IL-10 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-rat IL-13 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-rat TNF alpha antibody     -   11. 2400 uL Diluent 40 from Meso Scale Discovery (Rockville,         Md.)         For 1 plate of Panel 5, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-mouse IFN gamma antibody     -   2. 60 uL of 50× SULFO-TAG Anti-mouse IL-1 beta antibody     -   3. 60 uL of 50× SULFO-TAG Anti-mouse IL-2 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-mouse IL-4 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-mouse IL-5 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-mouse IP-6 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-mouse KC/GRO antibody     -   8. 60 uL of 50× SULFO-TAG Anti-mouse IL-10 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-mouse IL-12p70 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-mouse TNF alpha antibody     -   11. 2400 uL Diluent 45 from Meso Scale Discovery (Rockville,         Md.)

(vi) Preparation of Read Buffer

Read Buffer T (also available from Meso Scale Discovery) is obtained as a 4× stock solution and the working solution was 2×. For 1 plate, equal parts (10 mL) of Read Buffer T (4×) was combined with deionized water (10 mL). A working solution of read buffer was prepared in advance and stored at room temperature in a tightly sealed container (stable for up to three years).

(vii) Preparation of MSD Plate

Multi-well plates (also available from Meso Scale Discovery) were pre-coated with capture antibodies (FIG. 1) and exposed to a proprietary stabilizing treatment to ensure the integrity and stability of the immobilized antibodies. Plates were used as delivered; no additional preparation (e.g., pre-wetting) was required.

Example 2 Assay Protocol

(i) Fifty (50) uL of diluted sample (standards, controls, or unknowns) per well were added. The plate was sealed with an adhesive plate seal and incubated for 2 hours with vigorous shaking (300-1000 rpm) at room temperature.

(ii) The plate was washed 3 times with 150-300 uL/well of PBS-T. Twenty-five (25) uL of detection antibody solution was added to each well. The plate was sealed with an adhesive plate seal and incubated for 2 hours with vigorous shaking (300-1000 rpm) at room temperature.

(iii) The plate was washed 3 times with 150-300 uL/well of PBS-T. One hundred fifty (150) uL of 2× Read Buffer T (Meso Scale Discovery, Rockville, Md.) was added to each well. The plate was analyzed in a SECTOR® Imager (Meso Scale Discovery, Rockville, Md.).

Example 3 Panel Verification

Assay development and evaluation of assay performance was executed utilizing industry and regulatory guidelines. During product development, kit components and protocols were developed and optimized to yield optimum product performance. The robustness of the assay protocol was evaluated to examine the boundaries of selected incubation times. Accelerated stability studies for calibrators, antibodies, and controls were performed during assay development and were augmented with real-time stability studies on complete kits out to 36 months from the date of manufacture. Verification of product design specifications was performed by evaluating standard curves, and a set of controls for each panel (also obtained from Meso Scale Discovery, Rockville, Md.) for three days by two independent analysts for a total of eight plates. Each plate was considered as a run. A summary of the standard curve data is shown in FIGS. 2( a)-(e) and Tables 4-8.

Intra- and inter-run precision and accuracy for a set of controls for each panel was evaluated for nine runs. Precision and accuracy were verified for each lot as part of the lot verification and quality control release. The typical specification for precision is a concentration CV of less than 20% for controls on both intra- and inter-day runs. As part of product verification, the performance of each panel was evaluated for spike and recovery and dilution linearity in serum, heparin plasma, EDTA plasma, citrate plasma, CSF, urine, and/or cell culture supernates. Native human analyte levels were measured in serum, heparin plasma, EDTA plasma, citrate plasma, CSF, and urine. Native rat and mouse analyte levels were measured in serum, heparin plasma, EDTA plasma, and urine.

Pooled human blood was stimulated in vitro with different stimuli (LPS and Zymosan and Peptidoglycan) and at the end of the stimulation period, plasma was isolated. In addition, for panels 1-3, THP-1 cell line was stimulated with LPS and at the end of stimulation, lysates were prepared. Freshly isolated PBMC were treated with different stimulating agents and supernates were isolated. The plasma, the cell lysates, and PBMC supernates were then evaluated for native human analyte levels using panels 1-3. For panel 4, rat macrophase cell line NR8383 was stimulated with LPS, PHA, and Pokeweed mitogen (PWM) and the cell lysate and cell culture supernates were isolated. The plasma, cell lysates, and cell culture supernates were evaluated for native rat analyte levels using panel 4. For panel 5, RAW cell line was stimulated with LPS and J774A.1 cell line was stimulated with LPS and PWM and at the end of stimulation, lysates were prepared. The plasma and cell lysates were then evaluated for native mouse analyte levels using panel 5.

FIG. 2( a)-(e) shows a standard curve graph that illustrates the dynamic range of each panel (panels 1-5, respectively).

TABLE 4 Panel 1 Typical Data IFNγ IL-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 364 1.5 0

0.31 485 5.0 0.12 1435 5.2 1.2

5.1 0.49 2640 4.6 4.9 2120 2.7 2.0

4.0 20 7550 1.0 7.8 25 

3.5 78

 285 1.1 31 97 601 2.3 313 114 

2.0 125 305 

4.3 1250 420 

1.6 500 1 523 304 2.5 IL-2 IL-4 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0

12.0 0 203 11.1  0.31 533 9.0 0.05 365 10.1  1.2 1257 5.4 0.21 933 8.1 4.9 3026 5.7 0.82 3000 6.6 20  13 922 3.2 3.3  12 555 2.1 78  55 350 3.2 13  47 521 2.0 313 207 

2.0 53 178 863 2.5 1250 740 647 3.0 210 618 057 2.2 IL-6 IL-8 IL-10 Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 219 8.0 0 208 12.0 0 232 10.7  0.16 404 9.4 0.12 370 5.4

420 9.8 0.53 982 4.8 0.49 858 4.3 0.30 937 5.4 2.5

3.6 2.0 2724 2.7 1.2 2970 3.1 10

2.7 7.8 10 113 2.5 4.8 10 699 4.3 1 49 

3.4 31 41 493 2.2 19 43 376 3.5 163 215 238 3.7 125 169 

2.6 78 163 

2.1 650

2.7 520 722 

2.2 310 568 597 2.3 IL-12p70 IL-13 TNFα Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 267 15.5

100 10.5 0 138 19.2  0.10 327 13.1 0.11 241 15.1 0.03 266 12.8  0.41 511 7.2 0.48 405 9.3 0.32 606

1.6 1348 6.1 1.8 1030 3.7 1.3 1960 3.1 6.6 4787 3.6 7.3 3543 2.0 5.2

2.5

 18 351 2.3 20  17 020 2.1 21  29 253 2.6 105  69 571 6.3 118 11 0701 4.4

118 431 4.7 420 250 748 2.8 470 507 447 1.1 330

 866

indicates data missing or illegible when filed

TABLE 5 Panel 2 Typical Data GM-CSF IL-1α Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 221 14.3 0 401 11.9 0.24 355 7.5 0.1 531 7.5 0.98 781 5.5 0.4 852 5.8 3.9 2374 4.8 1.4 2167 8.4 16 9635 4.3 5.8 7368 5.1 63  35 827 3.4 23  27 075 5.5 250 139 828 3.4 93 110 306 3.2 1000 472 016 3.2 370 394 888 3.6 IL-5 IL-7 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 571 10.5 0 235 12.6 0.2 833 8.7 0.2 342 9.5 0.8 1438 7.4 0.7 712 6.1 3.1 3961 5.8 2.9 2107 5.1 12  14 364 7.9 12 8770 6.3 49  52 918 3.4 47  32 322 3.2 198 198 664 3.6 188 137 340 2.8 790 639 511 5.7 750 581 986 2.1 IL-12/IL-23 p40 IL-15 IL-16 Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 285 8.7 0 227 8.3 0 264 11.3 0.7 431 7.1 0.2 338 8.6 0.61 326 11.4 2.9 874 3.5 0.7 661 6.1 2.4 477 6.6 12 2531 3.8 2.7 1954 4.6 9.8 1090 4.8 47  10 105 6.5 11 7840 7.0 39 4481 6.4 188  36 783 5.9 44  28 139 3.5 156  15 741 2.1 750 144 847 2.8 175 105 824 5.3 625  82 935 4.1 3000 512 130 5.5 700 464 580 3.0 2500 436 497 4.5 IL-17A TNFβ VEGF Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 144 28.9 0 248 10.9 0 476 5.2 1.2 290 10.3 0.1 456 6.1 0.3 547 8.9 4.8 821 8.1 0.6 1156 3.8 1.0 681 5.4 19 2719 4.3 2.4 3813 3.0 4.2 1235 3.5 76  12 266 6.8 10 15143 2.4 17 4187 7.1 304  45 477 4.4 38  57 815 2.1 67  14 990 3.7 1216 190 122 2.5 153 233 155 1.3 268  93 227 3.4 4870 684 182 4.4 610 890 796 3.2 1070 517 033 3.1

TABLE 6 Panel 3 Typical Data Eotaxin MIP-1α Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 180 13.2 0 91

0.37 215 7.5 0.24 100

1.5 252 14.5 1.0 313 5.9 2.0 430 6.3 3.0 1033 3.9 23

5.5

5293 3.0

 21 595

 35 620 2.3 375

250 210 

3.4 1500 613 344 3.4 1000 834 

4.2 Eotaxin-3 TARC Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 114 35.7 0 283

1.2 208 10.7 0.37 546 8.5 4.9 501 5.3 1.5 1400 3.5 20

3.4 5.9 4580 3.0 78

23  18 048 4.4 313  23 154 3.0 94

2.7 1250 85 

  5.2 375 269 113 2.7 5000 283 993

1500 873 335 2.9 IL-10 MIP-1α IL-8 Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 58 29.3 0

18.3 0 127 21.5

435 7.1 0.2 170 16.9 18 218 23.5 2.4 1532 4.7 1.0 185 15.5 71 338 10.1 10

3.5 3.9 297 15.3 283

7.6

22 406

15 1023 5.1

4028 4.8 39 54 024 4.2

7812 2.7 4525 31 

7.4

3.7 248

4.2 18100 314 

4.6 2500

7.1

454 451 3.0 72400 1 784 204 3.3 MCP-1 MDC MCP-4 Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 171

0 152 10.3 0 71 29.0 0.1 277 11.1 2 200 6.0 0.2 121 26.7 0.5 654 6.6 10 677 3.3 0.6 120

2.0 22 406 6.4 39 2273 4.1 2.4 187 16.9 7.8

6.1 150 9996 3.0 10 976

4.1

59 

4.3

9027

125

3.3 2500

4.2 150

500

0.3 10000

3.0 625 494 077

indicates data missing or illegible when filed

TABLE 7 Panel 4 Typical Data IFNγ IL-2 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 462 11 0 334 13 1.2 663

12 330 16 4.9 1383 6.3 49 425

20 4056

195 747 5.9 78   15 075 2.7 781 1969 6.1 313   65 765

3125 7571 5.7 1250  316 965 2.3 12500

4.3 5000 1 334 697 4.5 50000 147 984 7.6 IL-4 IL-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 281 11 0 523 14 0.2 348 11 2.6 506 13 1.0 499 7.9 11 542 9.2 4.0 1184 6.3 42 683 7.6 16 3974 6.4 169 1508 2.9 64  21 355 4.2 675 4915 2.9 255 132 949 3.7 2700 20 813 2.8 1020 660 873 4.9 10800 93 359 4.4 IL-5 IL-6 KC/GRO Conc. Aver- Conc. Aver- Conc. Aver- (pg/ age % (pg/ age % (pg/ age % mL) Signal CV mL) Signal CV mL) Signal CV 0

14 0 491 7.4 0 648 9.9 2.4 220 15 3.0 485 8.8 0.7 687 5.6 9.38 250 9.0 12 542 7.2 2.7 714 6.0 39 400 9.0 48 729 9.0 11 781 5.4 156 1477 6.6 194 1565 5.8 43 1228 6.8 625  11 226 6.1 775 5186 5.7 174 5568 5.0 2500  75 782 4.3 3100  22 199 5.6 695  49 356 3.9 10000 295 607 6.3 12400 108 108 11 2780 387 251 6.6 IL-10 IL-13 TNFα Conc. Aver- Conc. Aver- Conc. Aver- (pg/ age % (pg/ age % (pg/ age % mL) Signal CV mL) Signal CV mL) Signal CV 0 596 14 0 275 16 0 208 17 4.9 583 20 0.4 269 17 0.3 259 9.9 20 658 16 1.6 336 9.1 1.1 404 10 78 1630 7.9 6.3 563 8.6 4.5 993 6.1 313 5131 6.7 25 1544 3.6 18 3323 4.6 1250 19039 5.2 100 7033 4.5 73  15 943 2.6 5000  73 865 4.3 400  44 979 2.3 290  92 423 3.7 20000 278 070 6.1 1600 299 022 6.2 1160 535 091 4.4

indicates data missing or illegible when filed

TABLE 8 Panel 5 Typical Data IFNγ IL-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 311 14.5 0 284 12.4 0.20 621 5.6 0.34 562 6.3 0.78 2443 2.4 1.4 1462 3.6 3.1 8641 2.3 5.5 4909 2.6 13  35 510 1.9 22 1 9260 2.1 50 137 358 1.8

7 7541 2.2 200

2.2 350   307 666 1.9 800 143 7412 2.2 1400 1 149 886 1.9 IL-2 IL-4 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0 289 12.0 0 485 6.9 0.51 513 6.2 0.34 732 7.3 2.1 1232 5.4 1.4 1529 4.0 8.2 4094 3.6 5.5 4351 2.9 33  15 136 3.5 22  17 081 1.6 131  64 545 3.4

 65 254 2.7 525 264 160 2.3 350 245 316 2.6 2100 939 169 1.6 1400 798 882 2.1 IL-5 IL-6 KC/GRO Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 145 21.6 0 369 7.8 0 307 10.3 0.2 412 10.6 1.1 544 6.0 0.39 480 9.1 0.75 1298 4.2 4.4 1107 5.9 1.6 1129 2.6 3.1 4826 2.6 18 3321 2.3 6.3 3695 1.9 13   19 027 3.0 70  12 162 1.6 25   13 975 2.8 50   79 736 3.9 781  50 520 2.6 100   65 482 2.6 200  336 576 2.9 1125  225 797 2.2 400  335 078 1.9 800 1 28 2609 2.9 4500 1 085 547 2.8 1600 1 609 664 2.1 IL-10 IL-12p70 TNFα Conc. Average Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV (pg/mL) Signal % CV 0 763 10.1 0 372 7.6 0 883 5.6 0.63 819 5.9 6.4 453 11.1 0.12 985 5.5 2.5 1125 6.2 26 684 5.1 0.49 1291 4.3 10 2349 3.1 103 1754 2.9 2 2592 3.1 41 7758 3.4 413 6261 2.6 7.8 7460 3.4 163  29 289 2.2 1650  30 330 2.9 31  29 633 2.9 650 121 581 3.7 6600

2.6 125 128 475 3.0 2600 504 569 4.3 26400 731 703 2.9 500 582 743 3.3

indicates data missing or illegible when filed

The lower limit of detection (LLOD) is a calculated concentration based on a signal 2.5 standard deviations above the background (zero calibrator blank). The LLOD shown in Tables 9-13 for each panel was calculated based on 8-9 runs.

TABLE 9 Panel 1 LLOD IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Median LLOD 0.22 0.05 0.12 0.01 0.07 0.01 0.01 0.13 0.27 0.04 (pg/mL) LLOD Range 0.18- 0.02- 0.01- 0.02-

0.03-

0.21-

(pg/mL) 0.37

0.13 0.04 1.0 0.01 0.05 0.19 0.41 0.10

indicates data missing or illegible when filed

TABLE 10 Panel 2 LLOD IL-12/ IL-23 GM-CSF IL-1α IL-5 IL-7 p40 IL-15 IL-16 IL-17A TMFβ VEGF Median LLOD 0.14 0.08 0.10 0.15 0.40 0.14 1.60 0.77 0.06 0.9 (pg/mL) LLOD Range 0.10- 0.05- 0.08- 0.11- 0.30- 0.08- 0.96 0.50- 0.04- 0.75- (pg/mL) 0.14 0.29 0.28 0.22 0.58 0.19 2.77 2.70 0.12 1.39

TABLE 11 Panel 3 LLOD Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Median LLOD 3.2 0.58 1.2 0.13 0.13 1.6

2.6 2.3 (pg/mL) LLOD Range

3.7-

(pg/mL)

0.99 3.1 0.32 0.22 2.3 62 0.38 2.8 2.7

indicates data missing or illegible when filed

TABLE 12 Panel 4 LLOD IFNγ IL-2 IL-4 IL-1β IL-5 IL-6 KC/GRO IL-10 IL-13 TNFα Median LLOD 0.7 57 0.7 35 27 23 21 14 3.7 0.9 (pg/mL) LLOD Range 0.4-2.7 38-126 0.4-1.0 16-80 19-37 16-41 19-30 8.9-20 2.7-8.6 0.5-1.5 (pg/mL)

TABLE 13 Panel 5 LLOD IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα Median LLOD 0.042 0.11 0.22 0.19 0.056 0.606 0.22 1.1 8.9 0.15 (pg/mL) LLOD Range 0.025- 0.093- 0.165- 0.099- 0.05- 0.486- 0.185- 0.518- 7.598- 0.109- (pg/mL) 0.084 0.171 0.338 0.343 0.099 1.075 0.373 3.19 14.254 0.548

Controls were made by spiking calibrator into non-human animal matrix for panels 1-3, rat serum for panel 4, and mouse serum for panel 5, at levels throughout the range of the assay. Analyte levels were measured using a minimum of 3 replicates on 3 runs over 3 days. Average intra-run % CV is the average % CV of the control replicates within an individual run. Inter-run % CV is the variability of controls across a selected number of runs. Inter-lot % CV is the variability of controls across a selected number of kit lots.

TABLE 14 Panel 1 Average Average Conc. Intra-run Inter-run Inter-lot Control Runs (pg/mL) % CV % CV % CV IFNγ High 9 1941 1.9 6.8 4.8 Mid 9 203 1.9 8.3 Low 9 16 4.8 7.8 IL-1β High 9 107 2.6 5.3 Mid 9 11 1.8 6.5 Low 9 7 3.4 9.8 IL-2 High 9 986 2.2 3.2 Mid 9 99 2.2 4.8 Low 9 9 4.9 13 IL-4 High 9 294 1.7 6.2 Mid 9 32 4.4 6.7 Low 9 4 3.5 5.8 IL-6 High 9 801 3.4 5.8 Mid 9 76 3.0 4.6 Low 9 6 4.1 7.0 IL-8 High 9 613 2.3 5.2 Mid 9 60 1.8 6.1 Low 9 8 3.4 8.4 IL-10 High 9 372 2.2 2.8 Mid 9 39 1.5 5.5 Low 9 4 3.3 8.6 IL-12p70 High 9 467 4.4 6.4 Mid 9 51 3.2 6.6 Low 9 5 3.9 4.3 IL-13 High 9 657 3.0 8.6 Mid 9 74 2.7 13.8 Low 9 5 6.3 11.6 TNFα High 9 270 4.0 7.2 Mid 9 24 3.7 9.6 Low 9 3 3.7 12.0

TABLE 15 Panel 2 Average Average Conc. Intra-run Inter-run Inter-lot Control Runs (pg/mL) % CV % CV % CV GM-CSF High 9 506 4.0 22.1 N/A Mid 9 53 2.6 20.8 N/A Low 9 5 4.8 16.8 N/A IL-1α High 9 144 3.4 11.8 N/A Mid 9 15 3.2 12.4 N/A Low 9 2 5.7 13.8 N/A IL-5 High 9 450 4.1 16.6 N/A Mid 9 45 2.4 18.2 N/A Low 9 4 4.4 16.0 N/A IL-7 High 9 437 3.3 9.7 N/A Mid 9 44 2.9 4.8 N/A Low 9 5 3.8 4.6 N/A IL-12/IL-23 p40 High 9 1631 3.7 10.9 N/A Mid 9 173 2.4 9.9 N/A Low 9 17 3.9 8.0 N/A IL-15 High 9 317 3.5 26.0 N/A Mid 9 35 3.8 31.1 N/A Low 9 4 4.7 25.6 N/A IL-16 High 9 1965 2.3 19.8 N/A Mid 9 166 1.7 23.7 N/A Low 9 19 5.2 25.8 N/A IL-17A High 9 2662 5.3 20.0 N/A Mid 9 256 4.3 18.1 N/A Low 9 25 4.3 16.2 N/A TMFβ High 9 298 4.4 27.2 N/A Mid 9 30 3.0 27.7 N/A Low 9 3 3.0 25.0 N/A VEGF High 9 766 2.6 18.3 N/A Mid 9 64 2.7 12.1 N/A Low 9 8 5.0 5.4 N/A

TABLE 16 Panel 3 Average Average Conc. Intra-run Inter-run Inter-lot Control Runs (pg/mL) % CV % CV % CV Eotaxin High 9

2.3 5.9 Mid 9 312 2.3 4.5 Low 9 34 12.8

MIP-1β High 9 2071 3.0

Mid 9 222 1.4

Low 9 20 4.4

Eotaxin-3 High 9 13931 2.5

Mid 9 1025 4.4

Low 9 131 7.0 9.5 TARC High 9 3240

6.2 Mid 9 332

3.6 Low 9 34 4.3

IP-10 High 9 5850

14.4 Mid 9 435 3.5

Low 9 51 4.2 10.2 MIP-1α High 9 2253 1.8 4.0 Mid 9

1.6

Low 9 26

IL-8 High 9 125226 2.1

Mid 9 44664 1.3 13.2 Low 9 4830 2.1

MCP-1 High 9 1066 5.1

Mid 9 113

4.4 Low 9 11 6.3 6.2 MDC High 9 25521 4.6

Mid 9 1549 4.1 5.2 Low 9 197 4.1 6.3 MCP-4 High 9

Mid 9 170 2.5

Low 9 14 12.1 14.6

indicates data missing or illegible when filed

TABLE 17 Panel 5 Average Average Conc. Intra-run Inter-run Inter-lot Control Runs (pg/mL) % CV % CV % CV IFNγ High 9 305 2.1 4.5 4.8 Mid 9 722 2.2 9.6 Low 9 23 1.3 6.6 IL-1β High 9 826 2.0 3.4 Mid 9 928 2.0 7.5 Low 9 53 1.7 5.2 IL-2 High 9 2,092 2.3 3.8 Mid 9 2,293 2.2 7.6 Low 9 60 2.4 5.5 IL-4 High 9 759 3.8 6.0 Mid 9 836 2.9 0.3 Low 9 70 2.4 6.8 IL-5 High 9 849 2.0 4.2 Mid 9 981 2.8 7.0 Low 9 36 2.2 4.8 IL-6 High 9 115 2.4 3.7 Mid 9 400 3.5 11 Low 9 26 2.5 5.4 KC/GRO High 9 776 3.1 3.4 Mid 9 752 2.7 6.2 Low 9 106 3.4 4.8 IL-10 High 9 3,370 3.1 4.3 Mid 9 4,167 3.1 7.6 Low 9 627 2.4 6.1 IL-12p70 High 9 7,621 4.7 7.8 Mid 9 26,735 7.0 9.9 Low 9 3,193 4.5 12 TNFα High 9 448 2.5 5.0 Mid 9 479 2.1 7.0 Low 9 22 3.0 5.1

To assess linearity in panels 1-3, normal individual human serum, EDTA plasma, heparin plasma, citrate plasma, and CSF samples from a commercial source were spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold before testing. Normal individual human urine was spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, and 16-fold. Percent recovery at each dilution was calculated by dividing the dilution adjusted calculated concentration by the expected concentration, i.e., the calculated dilution adjusted concentration at 2-fold dilution for panels 1-2 and a 4-fold dilution for panel 3 (see equation below).

To assess linearity in panel 4, normal rat serum, EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were spiked with recombinant calibrators and diluted 4-fold, 8-fold, 16-fold, and 32-fold before testing. Percent recovery at each dilution was calculated by dividing the dilution adjusted calculated concentration by the expected concentration, i.e., the calculated dilution adjusted concentration at 4-fold dilution.

To assess linearity in panel 5, normal mouse serum, EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold before testing. Percent recovery at each dilution was calculated by dividing the dilution adjusted calculated concentration by the expected concentration, i.e., the calculated dilution adjusted concentration at 2-fold dilution.

The average percent recovery shown below is based on samples within the quantitative range of the assay.

${\% \mspace{14mu} {Recovery}} = {\left( \frac{Measured}{Expected} \right)*100}$

TABLE 18 Panel 1 IFM IL-1 IL-2 IL-4 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 105  95-109 106 100-118 91 78-121 106 93-128 (N = 12) 8 101  91-112 103  92-129 91 71-158 103 97-133 16 100  92-119 102  85-121 94 63-196 107 94-139 32 98  87-120 105  85-136 107 63-283 103

64 102  88-125 110  88-143 120 63-402

EDTA 4 108 101-124 108 100-115 92 81-121 110 96-129 Plasma 8 107  93-131 106  94-119 91 75-157 108 86-140 (N = 12) 16 108  89-135 107  85-125 96 69-210 111

32 103  79-135 107  81-128 105 66-282 105

64 109  80-141 112  84-136

65-412 109 76-152 Reparin 4 106  87-116 109 100-123 94 76-122 107 92-128 Plasma 8 101  90-110 108  99-118 96 70-161 104 84-142 (N = 12) 16 102  89-112 106  93-122 102 65-206 108 82-151 32 98  84-112 108  98-124 110 61-277 105 81-149 64 101  83-124 109  93-137 125 54-435 110 91-157 Citrate 4 102  95-107 100  92-105 79 61-116 103

Plasma 8 97  87-104 99  94-107 74 50-146 99 89-110 (N = 10) 16 94  85-105 95  89-109 71 46-174

96-115 32 89  80-104 94  80-113 72 46-191 95 92-124 Urine 64 91  81-106 94  84-113 73 45-207 99 85-129 (N = 5) 4 96  92-100 98  93-102

75-103 97 93-102 8 88 79-91 91 85-96 79 63-96  98 90-110 16 87 83-90 90 88-94

58-100 99 90-112 Cell 4 102  95-105 100  95-105

101

Culture 8 97  92-103 96  90-104

78-88  100 94-107 Supernates 16 95  89-105 89  93-967 77 73-81  102 94-114 (N = 5) 32 88 82-94 85 81-99 75 71-77  95 87-103 64 90 80-97 85 75-84 70

94 78-104 IL-6 IL-8 IL-10 IL-12p70 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 105  95-113 97 89-103 102 93-108 104 99-117 (N = 12) 8 105  95-124 93 86-104 102 91-114 102

16 104  89-117

78-100 97 89-113 104 91-119 32 104  93-118 92 79-106 102 90-123 105 93-118 64 110  95-127 95 79-110 104 89-124 110 94-131 EDTA 4 104  99-115 97 94-104 106 100-116  106 94-126 Plasma 8 105  97-121 92 86-99  106 93-120 107 95-133 (N = 12) 16 106  90-132 90 74-104 103 82-119 108 92-142 32 105  88-133 90 71-102 107 81-131 108 87-152 64 113  93-144 95 72-108 106 81-132 114 88-154 Reparin 4

100-130 99 92-104 101 91-108 105 96-115 Plasma 8 105  84-121 94

101 85-111 102 94-116 (N = 12) 16 105  84-121 92 83-100 97 81-111 106 93-131 32 104

94 80-102 100 83-112 103 93-126 64 108  89-130

82-108 99 81-118 109 92-155 Citrate 4 107  92-169 97 93-107 97 94-100 101 90-115 Plasma 8 112

89 83-97  94 87-108 96 84-111 (N = 10) 16 127  85-416 85 74-95  88 74-105

78-109 32 135  78-550 85 72-97  90 75-114 91 73-111 Urine 64 156  79-702 85 73-99  88 71-111 95 74-118 (N = 5) 4 105 104-104 98 94-104 95 91-99  94 91-100 8 102 100-100 93 82-101 90 86-93  90 84-98  16 100 101-101 92 84-98  86 83-87  94 81-106 Cell 4  97

95 90-98  101 99-104 88 77-95  Culture 8  93  84-101 92

100 95-106 86 81-89  Supernates 16  87 79-95 85 80-92  94 87-100 84 81-89  (N = 5) 32  84 74-92

82-95  94 87-101 79 70-85  64  84 75-96 84 78-91  90 83-98  81 74-88  IL-13 TNF Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 4 88  79-103 98  86-107 (N = 12) 8 79  70-101 95  89-111 16 73  63-101 90  82-110 32 74  62-108 94  85-115 64 79  65-114 95  86-119 EDTA 4 90  84-102 97  93-103 Plasma 8 83  74-111 94  87-103 (N = 12) 16 77  62-117 89  77-103 32 76  53-117 92  77-104 64 81  59-127 94  76-107 Reparin 4 93  83-109 99  89-105 Plasma 8 84  72-114 96  81-101 (N = 12) 16 79

93  78-102 32 77  60-114 94 78-97 64 82  62-126 95  77-105 Citrate 4 87

95  90-101 Plasma 8 76

89  80-106 (N = 10) 16 66 57-78 83  70-108 32 65 57-79 84  71-107 Urine 64 67

84  71-108 (N = 5) 4 88

87 82-92 8 77 66-84 80 76-84 16 75 62-81 76 68-83 Cell 4 89 84-98 86 80-91 Culture 8 79 74-83 79 74-85 Supernates 16 70 65-73 72 63-81 (N = 5) 32 67 64-71 73 67-80 64

65-73

61-78

indicates data missing or illegible when filed

TABLE 19 Panel 2

IL-1α IL-5 IL-7 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 108  91-136 118 98-170 108 89-159 96  84-120 (N = 11) 8 93  77-141 120 65-220 101 76-134 85  65-104 16 94  68-145 138 59-320 94 74-129 82  64-107 32 89  64-144 175 63-621 99 73-129 78  66-108 64 92  66-143 209 75-834 99 76-125 82  71-122 EDTA 4 100  89-122 103 85-137 102 93-116 91 86-95 Plasma 8 91  80-123 101 78-182 98 82-124 82 79-91 (N = 11) 16 87  74-119 100 66-202 90 69-114 78 70-88 32 80  66-103 104 62-247

66-115 72 62-86 64 81  68-106 114 65-276 86 65-107 76 62-84 Reparin 4 102  83-135 105 88-139 106 87-123 98  84-106 Plasma 8 93  78-142 102 74-181 103 86-127 91 73-99 (N = 11) 16 93  72-152 109 63-258 96 77-127 89 72-99 32 89  72-144 114 59-294 97 73-124

64 93  74-159 134 66-422 96 65-122 93  66-110 Citrate 4 97 95-99 120 92-156 98 89-114 92  88-100 Plasma 8 85 81-88 129

92 78-115 82 73-94 (N = 10) 16 81 72-85 139 86-253

73-112 79 70-93 32 74 65-80 140 84-266 82 71-109 74 66-86 Urine 64 75 69-83 145 83-320 78 68-111 75 68-82 (N = 5) 4 114 104-122 131

108 95-132 107  95-112 8 122 104-127 116

121 111-134  111

16 131 127-135 102 91-127 132 124-155  124 114-132 Cell 4 93 86-98 110 95-124 94

88 85-91 Culture 8 91 86-98 109 96-137 89 87-92  89 83-93 Supernates 16 89  83-101 101 89-116 83

86 82-91 (N = 5) 32 88 83-95 105 96-122 83 81-85  89 82-97 64 91 84-99 104 88-120 80 78-83  92  86-100 IL-12/IL-23 p40 IL-15 IL-16 IL-17A Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 101  90-128 90  79-115 95  86-103 104  74-128 (N = 11) 8 91  65-114 85 69-94 88 72-99 95  58-108 16 90  65-114 80 62-94 86  73-101 90 64-95 32 87  71-107 83 73-98 91  77-101 87 72-96 64 89  78-119 83  70-101 98  83-122 88  77-100 EDTA 4 98  85-110 85 77-93 93  81-104 101  92-111 Plasma 8 91  72-106 82 75-94 79 68-93 96  86-108 (N = 11) 16 87  68-102 77 69-82 74 58-58 93  79-111 32 81 92-97 74 62-83 77  60-100 86  68-106 64 83 61-99 73 63-81 80  60-106 87  67-102 Reparin 4 107  91-133 83 72-93 97  79-115 102  83-110 Plasma 8 101  82-131 78 64-87 89

95  84-108 (N = 11) 16 103  80-145 75 64-84 87  57-107 94  82-104 32 99  73-130 74 58-89 93

87 76-97 64 103  78-144 75 61-85 99  63-123 89  75-109 Citrate 4 102  95-112 85 76-93 92

102  93-128 Plasma 8 96  77-113

70-90 81 76-91 94  81-143 (N = 10) 16 97  82-116 77

75 67-85 93  75-158 32 90  72-113 75 67-87 76 67-91 89  70-156 Urine 64 93  74-110 72 64-81 78 70-93 90  73-152 (N = 5) 4 116  50-142 107

134 118-123 113  95-148 8 127

120

140 127-183 124

16 140

131

135

Cell 4 103  94-109 80

101  95-110 99 83-91 Culture 8 95  90-101 80

99  95-103 81

Supernates 16 93  86-100 82

96  91-114 79 73-84 (N = 5) 32 90 87-94 84 77-90 107  97-129 79 73-84 64 94  90-100 90

112 102-140 84 76-89 TNF

Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 4 106 86-143 106  91-121 (N = 11) 8 100 76-116 95  77-113 16 98 72-116 96  70-122 32 99 69-120 118  74-170 64 97 71-116 145  82-213 EDTA 4 100 93-108 92  74-107 Plasma 8 94 83-108 83 69-96 (N = 11) 16 85 74-104 77 65-90 32 80 98-92  84  68-101 64 79

95  71-121 Reparin 4 102 90-114 107  85-115 Plasma 8 99 83-116 94  80-110 (N = 11) 16 96 82-114 82 69-99 32 94 77-111 96  57-130 64 94 77-114 139  54-234 Citrate 4 117

96  87-108 Plasma 8 122 102-159  89  77-102 (N = 10) 16 120 94-167 89  69-114 32 113 82-169 102  71-137 Urine 64 108 81-153 116  76-148 (N = 5) 4 111 94-119 106

8 120

16 143

112 105-119 Cell 4 87 86-89  89 87-90 Culture 8 84 82-86 

76-82 Supernates 16 81 79-83 

70-80 (N = 5) 32 81 79-83  76 72-82 64 82

77 72-82

indicates data missing or illegible when filed

TABLE 20 Panel 3

TARC Sample Fold Average % %

 Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 2 88  73-106 97  55-117 113  88-134 92  80-108 (N = 12) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 104 100-112 105  91-150 93  86-106 94  89-102 16 106  95-124 113  87-192 90  74-105 94  83-108 32 110  85-145 118  87-226 96  73-119 92  83-111 64 111  81-146 120  81-245 100  73-128 98  81-126 EDTA 2 91  84-102 93  59-107 131  95-163 94  79-109 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 104  85-115 108  99-143 78 61-96 95  85-106 16 105  92-119 114  96-177 72 59-93 95  78-112 32 105  87-135 123  96-207 73  60-103 88  72-127 64 105  85-151 122  91-220 77  64-115 92  76-131 Reparin 2 89  75-119 94  61-109 112  97-143 75  61-103 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 108 101-119 107  96-138 89  78-100 110  95-121 16 120  80-135 111  93-173 89  71-104 118  67-135 32 135  85-157 117  95-197 92  60-125 120  80-139 64 145  87-170 113  90-202 101  66-138 126  80-170 Citrate 2 95  85-104 106  99-117 122 111-137 97  89-141 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 10) 8 102  98-105 99  91-105 86 74-98 85  60-102 16 102  95-110 95  89-101 78 66-87 78 78-97 32 99  88-110 95  86-106 79 64-90 72 44-86 64 98  83-118 93  82-110 87  71-100 74 43-89 Urine 2 96  78-116 88  63-107 106  97-119 92  78-110 (N = 5) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 105 102-110 105 101-109 97  92-102 94  87-108 16 114 108-116 109 103-114 94 87-99 92  85-110 Cell 2 134 117-141 110  98-116 93 98-97 93  84-101 Culture 4 100 N/A 100 N/A 100 N/A 100 N/A Supernates 8 95  91-100 95  91-100 100  96-102 91 87-98 (N = 5) 16 95 88-99 94 90-99 108 101-114 87 80-94 32 95  89-101 91 87-99 112 105-118 82 75-89 64 99  88-107 95  89-102 126 116-133 90 76-99

Sample Fold Average

 Recovery Average

 Recovery Average

 Recovery Average

 Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 2 118 107-130 105  91-115 91  82-104 92 81-97 (N = 12) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 89 80-95 97  89-104 102  85-108 98  92-107 16 84 76-93 93  81-106 98  77-112 94  86-100 32 81 71-80 93  78-118 112  88-135 92  82-102 64 84 72-95 92  71-119 12  97-162 99  92-116 EDTA 2 117 104-149 103  73-112 93  84-108 99  93-107 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 89 81-96 99  90-111 90  79-100 92 83-97 16 86 73-99 97  88-113 87  75-100 88 78-96 32 86  71-100 98  86-116 98  83-109 88 77-98 64 91  75-106 97  80-119 112  94-127 95  83-108 Reparin 2 112  98-124 107 101-116 97  87-123 96  92-106 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 89 80-98 95  90-100 91  76-113 96  87-106 16 86 75-97 92  80-103 82 66-99 92  81-106 32 84  69-101 90  76-107 88  65-116 90  79-105 64 95  75-110 88  71-109 100  75-140 94  76-112 Citrate 2 131  98-169 109 102-115

 90-109 99  90-105 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 10) 8 88 72-97 94  88-105 88  79-100 91  83-101 16 82

87  78-108 79 73-90 87 82-95 32 79 62-89 83  74-111 87  79-102 83 74-89 64 84  65-101 79  70-108 97  87-114 86 78-94 Urine 2 93  75-102 111 104-115 117 107-126 95  91-101 (N = 5) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 97  93-106 96 95-99 87 78-94 98  96-102 16 97  87-109 92 89-97 77 68-35 96  91-103 Cell 2 179 127-256 120 104-128 89 84-94 98  93-108 Culture 4 100 N/A 100 N/A 100 N/A 100 N/A Supernates 8 73 55-84 90 85-84 86  89-113 95 89-98 (N = 5) 16 68 50-88 85 78-84 89 83-95 94  99-101 32 63 52-77 80 72-89 93 82-97 88 81-91 64 68 57-81 81 73-95 107  95-121 98  90-103

Sample Fold Average

 Recovery Average

 Recovery Type Dilution Recovery Range Recovery Range Serum 2 109 101-121 84 78-93 (N = 12) 4 100 N/A 100 N/A 8 89 81-96 113  98-127 16 79 66-87 116  97-141 32 72 57-83 125  99-168 64 69 48-81 123  97-174 EDTA 2 106  91-118 76 65-96 Plasma 4 100 N/A 100 N/A (N = 12) 8 93  83-101 115 102-144 16 84 69-94 122  97-163 32 79 65-89 126  89-188 64 76 64-87 125  83-193 Reparin 2 106  98-118 84 75-92 Plasma 4 100 N/A 100 N/A (N = 12) 8 90 78-99 112  99-125 16 78 62-89 118  94-154 32 73 57-83 124  87-181 64 70 57-82 127  96-209 Citrate 2 105  77-130 89 81-98 Plasma 4 100 N/A 100 N/A (N = 10) 8 88 69-94 102  99-108 16 74 97-85 96 90-06 32 68 55-75 94  86-103 64 64 52-69 95  86-110 Urine 2 92  71-112 96 95-97 (N = 5) 4 100 N/A 100 N/A 8 86  61-102 105 103-107 16 75 54-89 108 106-113 Cell 2 191 158-211 91 84-96 Culture 4 100 N/A 100 N/A Supernates 8 75 73-75 110 105-113 (N = 5) 16 63 61-65 113 105-120 32 56 54-59 111 103-118 64 55 52-57 113 105-124

indicates data missing or illegible when filed

TABLE 21 Panel 4 IFN IL-2 IL-4 IL-1β Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum  8 109% 100%-110% 100%  88%-107%  96%  91%-109% 106% 101%-119% (N = 5) 16 109%  87%-112% 103%  97%-109%  96%  88%-110% 118% 109%-126% 32 118% 106%-128% 102%  99%-106% 111% 106%-117% 125%  98%-163% EDTA  8 114% 100%-119% 102%  88%-105%  96%  92%-101% 112% 104%-120% Plasma 16 125% 107%-140% 110%  97%-124% 101%  97%-107% 135% 120%-1485 (N = 5) 32 127% 115%-136% 104%  96%-116% 105% 100%-112% 162% 102%-193% Reparin  8 116% 113%-119% 102% 100%-104% 112% 104%-119% 108%  97%-114% Plasma 16 130% 121%-147% 111% 101%-118% 118% 103%-124% 129% 115%-149% (N = 5) 32 141% 129%-162% 111% 104%-119% 131% 119%-139% 134% 111%-150% Urine  8 114% 100%-121% 102% 104%-105%  96%  92%-101% 112% 104%-120% (N = 5) 16 125% 107%-140% 110%  97%-124% 101%  97%-107% 135% 120%-148% 32 127% 115%-136% 104%  96%-116% 105% 100%-112% 162% 102%-193% Cell Culture  8 Supernates 16 (N = 4) 32 IL-5 IL-6 KC/GRO IL-10 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum  8 100%  99%-106% 100%  95%-107%  98%  95%-103% 100%  99%-106% (N = 5) 16 101%  87%-113% 102%  87%-111%  97%  87%-102% 101%  87%-113% 32 106%  96%-113% 117%  96%-135% 105%  93%-113% 106%  96%-113% EDTA  8  96%  83%-108% 107%  98%-125%  96%  93%-100%  96%  83%-108% Plasma 16 101%  92%-124% 119%  88%-149% 101%  94%-108% 101%  92%-124% (N = 5) 32  85% 62%-97% N/A N/A  93%  65%-106%  85% 92%-97% Reparin  8 106% 101%-116% 136% 118%-154% 116% 104%-128% 106% 101%-116% Plasma 16 110% 100%-127% N/A N/A 127% 116%-148% 110% 100%-127% (N = 5) 32 118% 107%-144% N/A N/A 131% 119%-155% 118% 107%-144% Urine  8  96%  83%-108% 107%  98%-125%  96%  93%-100%  96%  83%-108% (N = 5) 16 101%  92%-124% 119%  88%-149% 101%  94%-108% 101%  92%-124% 32  85% 62%-97% N/A N/A  93%  65%-106%  85% 62%-97% Cell Culture  8 Supernates 16 (N = 4) 32 IL-13 TNFα Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum  8 132% 126%-141% 103%  96%-112% (N = 5) 16 151% 140%-165% 108% 100%-115% 32 N/A N/A 121% 108%-132% EDTA  8 118% 111%-125% 104% 100%-107% Plasma 16 146% 144%-156% 113% 107%-121% (N = 5) 32 N/A N/A 117% 102%-126% Reparin  8 124% 117%-131% 114% 108%-119% Plasma 16 154% 136%-197% 123% 116%-129% (N = 5) 32 163% 150%-200% 139% 135%-146% Urine  8 118% 111%-125% 104% 100%-107% (N = 5) 16 146% 131%-156% 113% 104%-121% 32 N/A N/A 114% 102%-126% Cell Culture  8 118% 111%-125% 104% 100%-107% Supernates 16 146 131%-156% 113% 104%-121% (N = 5) 32 N/A N/A 117% 102%-126%

TABLE 22 Panel 5 IFN IL-1 IL-2 IL-4 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 107

106

184 120-154

8  95

 96

105

356 136-169 16

 90

 94 112-314 370 145-102 32

 99

371

64

105

304 144-204 EDTA 4 101

100

 95

123 144-120 Plasma 8 100

101

 94

134

16

 79-155 100

 90

32 103  79-124 100

 93

64 114  80-157 104

 94

Reparin 4 105  82-122 108

101

134

Plasma 8

 81-123 100

 95  90-104 147

16 107  95-171 100

32 109

100

 95

64

 57-147

 93

174

Citrate 4

220

Plasma 8  91  79-100  95  85-102  94

225

16

 71-103

 90 85-99

32

 75-104

64

 73-108

Urine 4  99

104

105

122

8  96

102

103

136

16  96

32  94

102

64

103

Cell 4 103 100-105 105 101-108  96  93-100 112 108-118 Culture 8  98 96-99 102  99-107  92 89-95 112 108-115 Supernates 16 100  96-104 102  99-108  88 87-90 115 112-122

32  95 94-97 101  97-106  92 91-93 112 109-116 64 100  96-105 104 101-107  93 87-96 119 111-125 IL-5 IL-6

IL-10 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4

118  105-154 320

8

90

317

16  99

90

106 

319

32  94

94

317

64

326

EDTA 4 100

97

91

103

Plasma 8  98  79-110 91

85

101

16  95  80-114 85

82

102

32  99  79-120 91

87

 90

64 105  82-133 95

90  72-112 102

Reparin 4 100  89-113 95

93  78-111 111

Plasma 8  95  87-105 95

107

16  93  85-110 97

85

108

32  93  78-121

105

64

 80-131 104 

Citrate 4

91

90

 99

Plasma 8

79

16

 70-102 79

 97

32

80

71

 83

64

82

74

Urine 4

99

95

103

8

94

70

16

 75-105 93

32

96

64  95

84

 95

Cell 4  95 94-98 93 91-94 76 72-80  93  87-100 Culture 8  93 92-97

88-92 69 63-72  84 79-92 Supernates 16  92 86-96 88 83-91 65 60-69  84 77-94

32  92 85-99 93 88-97 67 62-72  81 74-90 64  95  89-100 96  90-102 70 66-76  85  77-101 IL-12p70 TNFα Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 4

8

16

 97-323 32  79

64

EDTA 4  85

Plasma 8

100

16

 96

32

102  85-124 64

105

Reparin 4

102

Plasma 8

100

16  75

32  74

105

64

107

Citrate 4

Plasma 8

16

32

64

Urine 4

8 107

16 106

32 106

102

64 113

107

Cell 4  94 92-88  93 92-94 Culture 8  87 85-88  89 87-93 Supernates 16  83 79-88  89 84-93

32

78-87  93 88-96 64  85 79-93  96  92-100

indicates data missing or illegible when filed

Spike and recovery measurements of different sample types throughout the quantifiable range of the assays were evaluated. Multiple individual human serum, EDTA plasma, heparin plasma, citrate plasma, urine, and/or CSF samples from a commercial source and cell culture supernates were spiked with calibrators at three levels (high, mid, and low) and subsequently diluted two-fold.

TABLE 23 Panel 1 IFN IL-10 Spike Conc. Average % Spike Average % Sample Range % Recovery Level % Recovery Type (pg/mL) Recovery Range

Recovery Range Serum 17-20 100 91-126 6-7 88 81-99  (N = 12) 173-193  99 84-121 59-67 89 77-103 1835-1874 107 95-117 630-635 100  81-112 EDTA 17-17 101 84-114 6-6 96 64-119 Plasma 173-178 103 82-118 59-61 97 71-119 (N = 12) 1874-1902 103 78-127 635-665 97 71-125 Reparin 86-17

89-123 6-6 97 78-118 Plasma 171-178 102 85-121 61-62 94 72-114 (N = 12) 1871-1902  98 86-119 665-681 92 74-112 Citrate 16-20 103 94-113 5-7 97 71-117 Plasma 171-214 105 95-121 62-74 96 66-114 (N = 10) 1971-2108  97 91-109 681-735 90 72-104 Urine 19-20 101 94-108 7-7 96 88-107 (N = 50 204-214 103 93-111 69-74 99 90-108 2076-2108  99 91-106 733-735 95 83-101 Cell Culture 11-13 107 86-122 4-5 112  87-125 Supernates 168-173 105 97-123 60-61 115  101-133 

1919-2037  93 88-102 718-762 96 92-104 IL-2 IL-4 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 7-8 80 59-92 1-1 94 87-104 (N = 12) 63-73 83 63-96 7-7 97 90-107 725-768 97  83-119 74-76 109 94-118 EDTA 6-7 89  72-107 1-1 101 85-114 Plasma 63-66 91  72-109 7-7 104 83-114 (N = 12) 748-768 95  77-122 76-81 108 81-122 Reparin 6-7 90  49-107 1-1 98 87-115 Plasma 66-72 84  39-112 7-7 106 89-124 (N = 12) 748-881 89  47-111 78-81 103 89-119 Citrate 7-8 89   8-111 1-1 100 85-109 Plasma 72-79 92  11-120 7-8 108 93-119 (N = 10) 880-881 83   8-107 78-86 105 92-115 Urine 8-8 98  88-106 1-1 104 97-114 (N = 5) 79-81 95  82-104 8-8 102 93-110 880-908 91 74-99 84-86 100 84-107 Cell Culture 4-5 114  86-133 0-1 113 84-130 Supernates 66-67 114  98-139 7-7 116 103-138 

853-904 97  91-103 88-88 104 95-112 IL-6 IL-8 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 7-8 80 59-92 1-1 94 87-104 (N = 12) 63-73 83 63-96 7-7 97 90-107 725-738 97  83-119 74-76 109 94-118 EDTA 6-7 89  72-107 1-1 101 85-114 Plasma 63-66 91  72-109 7-7 104 83-114 (N = 12) 748-768 95  77-122 76-81 103 81-122 Reparin 6-7 90  49-107 1-1 98 87-115 Plasma 66-72 84  39-112 7-7 106 89-124 (N = 12) 748-881 89  47-111 78-81 103 89-119 Citrate 7-8 89   8-111 1-1 100 85-109 Plasma 72-79 92  11-120 7-8 108 93-119 (N = 10) 880-881 83   8-107 78-86 105 92-115 Urine 8-8 98  88-106 1-1 104 97-114 (N = 5) 79-81 95  82-104 8-8 102 93-110 880-908 91 74-99 84-86 100 84-107 Cell Culture 4-5 114  86-133 0-1 113 84-130 Supernates 66-67 114  98-139 7-7 116 103-138 

853-904 97  91-103 88-88 104 95-112 IL-10 IL-12p70 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 4-4 101 83-129 5-5 96 78-137 (N = 12) 39-44 101 91-123 47-53 99 73-158 408-433 113 100-123  495-499 100 85-133 EDTA 4-4 103 82-119 4-5 99 73-122 Plasma 39-41 102 81-117 47-48 97 75-118 (N = 12) 433-434 104 80-127 499-908 100 82-121 Reparin 4-4 105 90-124 4-4 103 78-126 Plasma 41-42 106 91-123 48-48 98 74-121 (N = 12) 434-466 103 91-122 506-519 99 70-122 Citrate 4-5 107 94-126 4-5 104 89-132 Plasma 42-50 106 94-126 78-59 100 83-127 (N = 10) 466-480 100 93-109 519-590 95 71-122 Urine 4-5 98 90-108 5-6 107 97-121 (N = 5) 48-50 98 87-108 57-59 107 92-117 480-480 94 79-104 550-556 104 89-121 Cell Culture 2-3 103 75-119 3-3 120 94-137 Supernates 38-42 104 90-123 46-49 111 93-135

422-487 93 86-99  522-547 111 103-119  IL-13 IL-TNFα Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 5-6 106  74-125 4-5 104  83-146 (N = 12) 45-50 118  77-205 40-45 107  84-166 552-564 125  89-139 457-460 110  79-125 EDTA 5-5 115  92-130 4-4 108  95-120 Plasma 45-46 120  88-142 40-42 112  96-125 (N = 12) 564-574 124  87-157 460-469 110  88-132 Reparin 5-5 111  81-134 4-4 111  99-131 Plasma 46-46 116  87-138 42-44 111 102-130 (N = 12) 574-595 114  81-128 459-509 108  96-122 Citrate 5-6 120 103-134 4-5 112 101-122 Plasma 46-55 127 111-149 44-41 114 102-131 (N = 10) 596-629 118 105-137 509-530 106  95-122 Urine 6-6 114 106-127 5-5 99  83-112 (N = 5) 55-55 124 117-132 81-51 103  84-119 629-633 119  98-127 530-539 110  91-120 Cell Culture 4-4 124  95-143 4-4 125  99-147 Supernates 46-54 135 116-169 47-54 127 114-154

617-695 118 104-126 581-672 117 104-127

indicates data missing or illegible when filed

TABLE 24 Panel 2

IL-1α Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 6-6 92  70-102 6-6 74  44-102 (N = 12) 60-61 91  66-104 60-69 70  22-107 563-580 90  65-108 623-626 71 18-90 EDTA 6-6 97  62-115 6-6 84  48-100 Plasma 60-60 96  62-120 60-63 88  24-112 (N = 12) 562-563 95  60-128 611-623 86  17-108 Reparin 6-6 91  51-120 6-6 69  11-105 Plasma 58-60 91  64-127 59-63 69  14-110 (N = 12) 506-582 94  64-123 555-611 69  12-108 Citrate 6-6 109  97-118 6-7 69 27-94 Plasma 58-59 104  96-110 59-64 63  23-100 (N = 10) 506-507 112  98-132 550-556 64  22-101 Urine 6-6 120 112-129 7-7 94 91-98 (N = 5) 57-59 120 106-138 61-64 102  90-116 507-515 126 113-132 543-550 104  90-113 Cell Culture   11 101  93-114   4 97  91-105 Supernates  105 95  87-103  34 90 76-96

1121 95  93-110 343 89 77-96 IL-5 IL-7 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 3-3 87 74-110 1-1 101  68-111 (N = 12) 31-32 87 63-107 12-13 100  74-112 300-332 85 54-105 124-131 100  83-117 EDTA 3-3 85 56-105 1-1 102  92-115 Plasma 31-35 85 57-101 12-12 106  92-119 (N = 12) 325-332 88 52-120 124-127 105  91-130 Reparin 3-3 84 57-107 1-1 87  76-113 Plasma 34-35 82 55-104 12-13 83  72-126 (N = 12) 326-351 86 61-112 126-127 88  75-124 Citrate 3-4 101 78-125 1-1 104  94-117 Plasma 34-35 101 75-125 13-13 111  97-120 (N = 10) 321-361 98 79-117 126-127 115  88-130 Urine 3-4 104 99-111 1-1 110 100-117 (N = 5) 32-35 108 105-114  13-13 111 97-125 305-321 114 109-125  125-127 120 108-135 Cell Culture   9 107 97-119   6 84 71-93 Supermates  86 99 90-105  66 80 74-86

838 105 88-130 691 84 77-93 IL-12/IL-23 p40 IL-15 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 21-21 92  81-104 3-4 98  78-110 (N = 12) 194-221 90  79-101 35-36 100  75-118 1819-2112 94  78-107 284-300 119  92-138 EDTA 21-21 91 81-99 4-4 100  92-110 Plasma 194-199 97  88-109 34-35 108  97-120 (N = 12) 1819-1951 96  79-130 290-300 122 103-143 Reparin 19-21 89 72-99 4-4 92  78-112 Plasma 193-199 86  71-121 34-35 103  88-132 (N = 12) 1684-1951 88  74-127 261-290 123 100-150 Citrate 19-20 91 85-99 4-4 104  87-113 Plasma 188-193 89  77-105 34-35 118 102-131 (N = 10) 1645-1684 95  71-117 261-278 150 129-173 Urine 20-20 127 119-131 3-4 105  98-113 (N = 5) 188-204 121 102-140 33-35 120 111-131 1645-1758 125 118-135 276-278 141 126-152 Cell Culture   38 119 104-132   6 76 71-89 Supernates  346 112  96-125  68 71 68-78

3784 110 105-143 593 85 71-95 IL-16 IL-17A Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 17-17 90 76-103 7-7 102  82-132 (N = 11) 142-146 94 69-104 63-70 103  88-129 1594-1627 90 63-108 682-833 91  77-124 EDTA 17-17 88 71-105 7-7 97  81-110 Plasma 142-153 98 68-105 63-69 102  84-120 (N = 11) 1627-1665 96 67-110 682-721 95  80-120 Reparin 17-18 90 64-128 7-7 97  60-145 Plasma 153-155 92 69-127 69-72 97  72-115 (N = 11) 1665-1713 90 66-124 703-721 96  78-113 Citrate 18-18 89 44-90  7-9 96  56-113 Plasma 155-156 100 42-81  72-79 99  59-117 (N = 10) 1679-1713 104 44-87  667-703 97  49-113 Urine 18-19 78 123-148 8-9 131 119-142 (N = 5) 153-156 75 128-152 74-79 142 126-171 1673-1679 68 128-149 667-711 134 105-155 Cell Culture   31 102 91-132    53 111  91-119 Supernates  387 85 89-126   561 117  96-128

3311 101 88-130  6298 111 108-133

Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 9-9 88  76-103 10-11 81 61-96

85-93 84  69-104 112-119 73  37-103 882-884 85  63-108 1708-1733 65  40-104 EDTA 9-9 93  71-105 11-12 88  80-115 Plasma 85-91 92  68-105 119-120 91  82-110

884-904 89  67-110 1706-1733 88  74-101 Reparin 8-9 89  64-128 12-17 78 39-96 Plasma 85-91 89  69-127 120-142 62  39-118

798-904 88  66-124 1676-1706 47  32-110 Citrate 8-8 70 44-90 17-19 71 35-96 Plasma 85-85 57 42-81 142-158 83  67-121 (N = 10) 784-798 69 44-87 1676-1739 78  58-113 Urine 8-8 135 123-148 18-19 94 88-98 (N = 5) 83-85 138 128-152 153-158 118 107-134 784-816 139 128-149 1739-1749 119 111-124 Cell Culture   6 117  91-132   12 92  85-100 Supernates  65 111  89-126  118 93  88-102

644 119  88-130 1718 140 131-151

indicates data missing or illegible when filed

TABLE 25 Panel 3

Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 70-75 100 86-113 51-54 107 72-123 (N = 12) 295-304 100 79-124 210-213 106 57-119 2336-2467 100 84-135 1988-2256 103 47-127 EDTA 74-75 99 95-114 54-59 106 68-120 Plasma 295-312 103 96-113 213-228 102 57-114 (N = 12) 2336-2483 113 96-129 1988-2114 100 56-119 Reparin 73-74 109 74-126 58-59 103 71-117 Plasma 312-331 91 68-106 228-241 97 51-109 (N = 12) 2345-2483 79 64-104 1866-2114 99 50-116 Citrate 73-73 100 82-113 58-59 108 102-121  Plasma 331-338 95 75-103 241-263 102 92-109 (N = 10) 2345-2483 99 76-119 1666-2203 107 93-121 Urine 73-73 111 97-119 55-59 114 109-121  (N = 5) 331-338 104 94-116 250-253 104 96-109 2398 101 75-125 2016-2203 102 89-115 Cell Culture 70-75 100 86-113 51-54 107 72-123 Supernates 295-304 100 79-124 210-213 106 57-119

2336-2467 100 84-135 1988-2256 103 47-127

TARC Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 250-271 105  83-135 65-74 106 71-123 (N = 12) 1044-1189 100  78-135 269-270 112 78-133 12348-12607 106  87-164 2719-2753 120 99-153 EDTA 271-279 126 114-135 74-79 96 69-105 Plasma 1044-1116 136 119-150 269-279 105 79-119 (N = 12) 12348-13154 146 102-177 2719-2969 106 77-127 Reparin 279-284 105  90-129 70-79 104 44-140 Plasma 1116-1257 105  92-128 279-287 87 46-111 (N = 12) 11390-13154 130  85-171 2592-2568 91 59-111 Citrate 273-284 110  93-126 70-70 105 94-121 Plasma 1257-1310 104  93-114 287-308 108 94-123 (N = 10) 11390-11682 112  98-130 2592-2692 124 100-226  Urine 273-298 113 109-119 70-82 129 98-162 (N = 5) 1310-1356 96  92-100 308-337 115 83-141 11682-11892 96  89-103 2692-2742 112 83-140 Cell Culture 262-297 91 84-95 81-89 119 115-126  Supernates 1161-1276 94 90-99 358-375 120 117-123 

 9225-10407 101  55-108 3194-3905 125 110-135 

Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 126-130 101  91-110 52-55 105  88-117 (N = 12) 457-522 111 102-120 213-215 110  90-125 5723-6188 113  79-131 1922-2000 112  91-123 EDTA 126-138 104  99-113 55-56 103  85-113 Plasma 475-511 115 103-124 213-218 112  95-125 (N = 12) 5982-6188 112  91-134 1922-2009 114  92-123 Reparin 138-145 99  92-104 55-56 97  80-106 Plasma 511-563 105  96-114 218-241 103  77-115 (N = 12) 5982-6172 117  77-138 1781-2009 113  77-130 Citrate 144-145 103  91-121 55-55 105  65-141 Plasma 563-632 102  87-113 241-22 104  67-115 (N = 10) 6172-6601 110  88-139 1781-1916 119  78-133 Urine 144-153 111 104-124 54-55 116 104-127 (N = 5) 566-632 102  97-108 242-242 115 109-123 6492-6601 84 55-97 1916-2167 121 116-129 Cell Culture 116-144 110  97-121 44-52 112 100-118 Supernates 648-681 134 121-150 230-259 117 111-124

 5276-28154 274 113-603 2341-2692 119 110-127 IL-8

Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 3826-3911 87 76-97 22-24 97  87-107 (N = 12) 12490-12841 91  81-110 91-92 102  90-109 156805-157779 88 77-99 916-928 100  77-109 EDTA 3911-4338 94  86-100 24-26 88 77-99 Plasma 12490-13720 104  81-115 91-96 90 84-96 (N = 12) 153349-156805 94  74-110 928-972 85  72-102 Reparin 3881-4338 84 73-93 26-27 102  88-130 Plasma 13720-14211 88 78-97  94-103 105  94-123 (N = 12) 143226-153349 93  71-102 877-972 100  80-116 Citrate 3727-3881 98  78-109 26-27 94  85-108 Plasma 14211-14529 94  82-102 103-108 95  85-108 (N = 10) 143225-144105 102  92-115 877-954 92  91-110 Urine 3727-3913 114 108-119 26-27 103  95-116 (N = 5) 13811-14529 105 101-108 106-108 105  96-117 144105-159737 105 103-106 924-954 102  90-112 Cell Culture 3697-4844 86 71-93 21-24 101  96-109 Supernates 17099-19472 103  95-109  98-105 106 103-110

167913-183991 103  97-106 994-1187 106  99-119

Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 421-443 97  86-115 31-33 104  70-124 (N = 12) 1707-1780 122 113-129 123-133 94  57-108 23795-24625 120 115-127 1016-1031 95  71-104 EDTA 443-465 99  91-105 31-32 107  78-127 Plasma 1707-1744 115 107-123 123-136 90  57-106 (N = 12) 23795-26159 113 101-128 1016-1085 89  64-101 Reparin 439-465 104  94-117 32-32 128 113-146 Plasma 1744-1948 114 101-130 136-137 108  86-121 (N = 12) 21657-26159 105  92-119  960-1085 94  72-111 Citrate 426-439 105  93-126 31-32 118 104-148 Plasma 1948-2048 111 106-125 137-147 97  85-107 (N = 10) 21657-23766 117  99-151  960-1075 102  90-111 Urine 426-465 130 112-140 30-31 125 118-134 (N = 5) 1967-2048 123  96-138 146-147 107  96-115 22796-23766 124  99-140 1057-1075 107 101-118 Cell Culture 527-592 129 122-137 16-24 87 64-93 Supernates 2694-2964 142 138-152 125-132 98  95-100

33418-39552 147 134-159 1120-1201 103 100-107

indicates data missing or illegible when filed

TABLE 26 Panel 4

IL-2 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 833.3  92%  84%-102% 8333.3  90%  79%-101%

206.3  88% 82%-96% 2083.3  95%  81%-108% 52.1  89% 80%-96% 520.8 102%  82%-113% EDTA 833.3  89% 86%-99% 8333.3  95%  89%-106% Plasma 206.3  89% 68%-98% 2083.3 104%  96%-110%

52.1  94%  86%-101% 520.8 109% 102%-115% Reparin 833.3  71% 66%-75% 8333.3  95%  91%-106% Plasma 208.3  70% 68%-71% 2083.3 103%  99%-105%

52.1  63% 57%-68% 520.8 111% 104%-118% Urine 833.3  88% 76%-96% 8333.3  64% 42%-56%

208.3  93%  74%-103% 2083.3  65% 43%-55% 52.1 100%  88%-108% 520.5  71% 72%-92% Cell Culture Supernates

Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum

95% 91%-102% 1800 59%  51%-65%

42.5 84% 80%-85%  450 65%  62%-72% 10.6 91% 90%-94%  112.5 77%  62%-93% EDTA 170 102%  99%-107% 1800 69%  65%-72% Plasma 42.5 94% 91%-104% 450 80%  74%-86%

10.6 103%  97%-105% 112.5 87%  82%-94% Reparin 170 91% 82%-95%  1800 75%  69%-81% Plasma 42.5 80% 69%-90%  450 82%  73%-88%

10.6 87% 74%-95%  112.5 87%  77%-104% Urine 170 75% 43%-101% 1800  265% 166%-389%

42.5 65% 41%-85%  450  183% 102%-253% 10.6 71% 47%-85%  112.5  161%  91%-235% Cell Culture Supernates

IL-5 IL-6 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 1665.7 87% 80%-97%  2065.7  95%  83%-108%

416.7 85% 82%-90%  515.7 107%  83%-116% 129.2 83% 67%-100% 129.2 109%  89%-127% EDTA 115.7 81% 69%-91%  2065.7  97%  88%-111% Plasma 416.7 84% 71%-93%  516.7 113%  96%-139%

129.2 91% 81%-100% 129.2 126% 111%-136% Reparin 1665.7 91% 81%-110% 2065.7 106%  99%-112% Plasma 416.7 92% 81%-114% 516.7 117% 105%-130%

1129.2 81% 55%-92%  129.2 128% 111%-154% Urine 1665.7 77% 32%-132% 2065.7 130% 124%-138%

416.7 80% 44%-118% 516.7 141% 126%-165% 129.2 90% 52%-140% 129.2 147% 118%-201% Cell Culture Supernates

IL-10 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 463.3 84% 68%-96% 3333.3  91%  80%-102%

115.8 90% 87%-98% 833.3  94% 83%-99% 29 99%  94%-109% 208.3  99%  86%-112% EDTA 463.3 88% 76%-95% 3333.3  98%  86%-106% Plasma 115.8 92%  76%-101% 833.3 113% 104%-125%

29 122%   94%-134% 208.3 117% 110%-130% Reparin 463.3 59% 41%-81% 3333.3  82% 66%-96% Plasma 115.8 61% 42%-77% 833.3  79% 70%-88%

29 76% 51%-99% 208.3  75% 67%-83% Urine 463.3 142%  132%-170% 3333.3 126% 118%-135%

115.8 130%  119%-157% 833.3 119% 104%-139% 29 120%  107%-135% 208.3 119% 100%-135% Cell Culture Supernates

IL-13 TNFα Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum 266.7 80% 66%-88% 193.3 87% 81%-92%

66.7 71% 67%-75% 48.3 83% 79%-87% 16.7 77% 65%-87% 12.1 100%   83%-112% EDTA 266.7 92% 89%-95% 193.3 85% 82%-93% Plasma 66.7 89% 86%-95% 48.3 84% 79%-91%

16.7 105%  102%-114% 12.1 91% 87%-95% Reparin 266.7 82% 73%-90% 193.3 81% 72%-85% Plasma 66.7 63% 58%-68% 48.3 73% 69%-77%

16.7 70% 58%-92% 12.1 84%  66%-107% Urine 266.7 82%  83%-121% 193.3 82% 46%-97%

66.7 83%  74%-110% 48.3 83% 52%-92% 16.7 104%   86%-121% 12.1 104%   62%-111% Cell Culture Supernates

indicates data missing or illegible when filed

TABLE 27 Panel 5 IFN IL-10 Average % Average % Sample Spike Level % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum High

117 100-120 High

106  94-110 (N = 5) Mid

117

Mid

106  81-121 Low

112  96-123 Low

103  81-112 EDTA High

 67-142 High

104  94-155 Plasma Mid

103-120 Mid

105  95-117 (N = 7) Low

 65-100 Low

Reparin High

105  65-100 High

 98 67-104 Plasma Mid

105

Mid

107 95-117 (N = 7) Low

Low

 94 82-102 Citrate High

115 100-131 High

 99 93-100 Plasma Mid

110  51-126 Mid

101 85-121 (N = 5) Low

102

Low

105 85-123 Urine High

102

High

(N = 5) Mid

105

Mid

Low

105

Low

Cell Culture High

102  96-111 High

102  83-103 Supernates Mid

111 111-112 Mid

104 102-126 (N = 4) Low

105

Low

105 89-104 IL-2 IL-4 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum High

108  94-115 High

Mid

105  94-117 Mid

 53-77 Low

105  94-113 Low

 53-01 EDTA High

103-117 High

Plasma Mid

Mid

  Low

Low

Reparin High

High

Plasma Mid

Mid

Low

Low

Citrate High

100

High

Plasma Mid

 99

Mid

Low

 99

Low

Urine High

High

Mid

Mid

Low

Low

Cell Culture High

High

Supernates Mid

105-111 Mid

104 103-105

Low

Low

IL-5 IL-6 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum High

111 101-121 High

112

Mid

108  92-122 High

105

Low

103  95-107 High

105

EDTA High

 83-126 High

117

Plasma Mid

High

108

  Low

Mid

Reparin High

 79-115 Low

Plasma Mid

102

Mid

104

Low

Low

Citrate High

104  90-115 High

102

Plasma Mid

103  91-114 Mid

 90

Low

 91

Low

Urine High

High

Mid

Mid

Low

100

Low

Cell Culture High

100-107 High

100 103-114 Supernates Mid

111 108-117 Mid

111 105-113

Low

 93-104 Low

100

KC/GRO IL-10 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum High

 84

High

Mid

 82

Mid

low

 85

Low

 89

EDTA High

101

High

Plasma Mid

Mid

  Low

Low

Reparin High

107

High

Plasma Mid

Mid

Low

Low

Citrate High

112

High

90

Plasma Mid

111  90-127 Mid

 84-100

Low

100  91-112 Low

 89

Urine High

High

Mid

Mid

Low

122-164 Low

Cell Culture High

122-120 High

112

Supernates Mid

135 125-141 Mid

121 118-122

Low

132 122-138 Low

125 122-127 KC/GRO IL-10 Average % Average % Sample Spike % Recovery Spike Level % Recovery Type

Recovery Range

Recovery Range Serum High

120 119-144 High

90 65-94

Mid

126 107-139 Mid

92

low

110 111-124 low

92 82-95 EDTA High

162

High

95

Plasma Mid

123 118-124 Mid

93

  Low

Low

91

Reparin High

High

Plasma Mid

Mid

95

Low

117

Low

91

Citrate High

100

High

94

Plasma Mid

Mid

94

Low

121

Low

96 91-96 Urine High

High

74

Mid

Mid

Low

Low

Cell Culture High

116 112-123 High

113 105-121 Supernates Mid

120

Mid

117 113-121

Low

122

Low

112

indicates data missing or illegible when filed

To assess specificity of the individual assays, each panel was run using blended antibodies with individual calibrators at concentration that yield signal around 100,000 counts.

${{Non}\text{-}{specificity}\mspace{11mu} (\%)} = {\left( \frac{{Specific}\mspace{14mu} {Signal}}{{Non}\text{-}{specific}\mspace{14mu} {Signal}} \right)*100}$

TABLE 28 Panel 1 Calibrator

IL-1 IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNF Concentration 157 34

45 120 50 108

208 325 (pg/mL) Highest Non- 0.01 0.01

0.02

0.15 0.01 0.05

Specificity %

indicates data missing or illegible when filed

TABLE 29 Panel 2 Calibrator

IL-5 IL-7 IL-12/

IL-15 IL-16 IL-17A TNF

Concentration 123 50 73 92 328

(pg/mL) Highest Non-

0.12

0.05 0.20 0.01 0.10 0.12

Specificity %

indicates data missing or illegible when filed

TABLE 30 Panel 3 Calibrator

TARC

MCP-1 MDC MCP-4 Concentration 250 100 1250 120

150

120

236 (pg/mL) Highest Non- 0.02

0.04

0.04 0.04 0.05 Specificity %

indicates data missing or illegible when filed

TABLE 31 Panel 4 Calibrator IFN

IL-2 IL-4 IL-1

IL-5 IL-6 BC/GR

IL-10 IL-13 TNFα Concen- 1250

5000 300 13000 2000

0 750 200 tration (pg/mL) Highest 0.05 0.08 0.00 0.00 0.14 0.35 0.34 0.4

0.81 0

Non- specificity (%)

indicates data missing or illegible when filed

TABLE 32 Panel 5 Calibrator IFN

IL-1

IL-2 IL-4 IL-5 IL-6 BC/GR

IL-10 IL-12p70 TNFα Concentration 40 10

160 300 60 420 200

00

010

(pg/mL) Highest Non- 0.01

0.02 0.02 0.04

0.19 0.46 0.07 0.03 specificity (%)

indicates data missing or illegible when filed

To assess the specificity of each antibody, each panel was run using blended calibrators with concentrations listed above and individual antibodies at 1× concentration.

TABLE 33 Panel 1 Antibody IFN

IL-1

IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Highest Non- 0.02 0.02 0.03 0.02 0.52 0.11 0.

0.43 specificity (%)

indicates data missing or illegible when filed

TABLE 34 Panel 2 Antibody IL-12/IL- C

-CSF IL-1

IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TN

VEGF Highest Non- 0.10 0.10 0.05 0.06 0.19 0.27 0.47 0.12

0.02 specificity (%)

indicates data missing or illegible when filed

TABLE 35 Panel 3 Antibody Eotaxin IMP-1

Eotaxin-3 TARC IF-10 MIP-1α MCP-1 MDC MCP-4 Highest Non- 0.05 0.29 0.20 0.12 0.76

0.49 0.75 1.2 specificity (%)

indicates data missing or illegible when filed

TABLE 36 Panel 4 Antibody IFN

IL-2 IL-4 IL-1

IL-5 IL-6 MC/GR

IL-10 IL-13 TNFα Highest Non- 0.00 0.1 0.0

0.12 0.22 0.11 0.25 0.06 specificity (%)

indicates data missing or illegible when filed

TABLE 37 Panel 5 Antibody IFN

IL-1

IL-2 IL-4 IL-5 IL-6 MC/GR

IL-10 IL-12p70 TNFα Highest Non- 0.01

0.01 0.05 0.02 0.02 0.05 0.04

0.04 specificity (%)

indicates data missing or illegible when filed

To evaluate the specificity of the Panel 1 Kit assays against other biomarkers, each kit was run using blended antibodies with individual recombinant human proteins.

TABLE 38 Panel 1 Protein IL-12/IL- IL-5 GM-CBF IL-1α IL-7 28 p40 IL-15 IL-18 IL-17A TNFα VEGF Concen- 40 63 23 47 188 44 156 304 38 57 tration (pg/mL) Highest  0.15  0.28  1  0.25  0.43  0.42  0.78  0.29  0.15  0.91 non- specificity (%) Protein Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Concen- 94 63 313 94 158 62 31 625 39 tration (pg/mL) Highest  0.71  0.48  0.45  0.17  0.24  1  0.57  0.27  1 non- specificity (%)

TABLE 39 Panel 2 Protein IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-13 TNFα Concentration 78 31 78 13 41 4 19 29 21 (pg/mL) Highest non-  0.31  0.23  0.16  0.23  0.26 0.26  0.27  0.68  0.37 specificity (%) Protein Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Concentration 94 53 313 94 156 52 31 525

(pg/mL) Highest non-  0.3  0.57  0.32  0.14  0.11  0.85  0.25  0.19  .92 specificity (%)

indicates data missing or illegible when filed

TABLE 40 Panel 3 Protein IL-12/L-28 IL-5 GN-CSF IL-1α IL-7 p40 IL-15 IL-14 IL-17A TMFβ VEGF Concen- 49 63 23 47

44 156

19 67 tration (pg/mL) Highest  0.19  0.10  0.34  0.22 0.26  0.30  0.49 0.10  0.10  0.41 non- specificity (%) Protein IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 THFα Concen- 38 31 79 13 43 4 19 26 29 21 tration (pg/mL) Highest  0.26  0.02  0.10  0.11  0.19

 0.39  0.61  0.31  0.23 non- specificity (%)

indicates data missing or illegible when filed

To evaluate the impact of multiplexing on assay signal, standards in the quantifiable ranges were compared between individual assays (individual calibrator and individual antibody) and multiplexed assays (blended calibrators and blended antibodies) using each kit. The calculated % signal difference between individual and multiplexed assay is shown below.

TABLE 41 Panel 1 Assay IFN

IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNP

Signal 0.02 0.02

0.02 0.52 0.11 1.05 0.11 0.04 1.43 Difference (%)

indicates data missing or illegible when filed

TABLE 42 Panel 2 Assays IL-12/IL- GM-CSF IL-1α IL-5 IL-7 23 p40 IL-15 IL-14 IL-17A THFβ VEGF Signal 4 30 24 27 2

72 14 3

Difference (%)

indicates data missing or illegible when filed

TABLE 43 Panel 3 Assay Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Signal 4 4 24 11 22 2 7

6 11 Difference (%)

indicates data missing or illegible when filed

TABLE 44 Panel 4 Antibody IFN

IL-2 IL-4 IL-1β IL-5 IL-6 MC/GRD IL-10 IL-15 TN

Signal 11

13 23

3 3 5

Difference (%)

indicates data missing or illegible when filed

TABLE 45 Panel 5 Assay IFN

IL-1β IL-2 IL-4 IL-5 IL-6 MC/GRD IL-10 IL-12p70 TN

Signal 14

2 16 15

7

1 17 Difference (%)

indicates data missing or illegible when filed

The kits were designed to minimize interference by receptors and other related proteins. For each panel, a multi-analyte calibrator in diluent and normal human were spiked with three different concentrations of receptors and binding partners. The recovered calibrator concentrations were compared to unspiked standards and normal serum.

All the assays in each panel were calibrated against a reference calibrator obtained from Meso Scale Discovery (Rockville, Md.). The NIBSC/WHO Standards for the following human analytes were evaluated against the MSD reference calibrators. To convert sample values obtained with a panel to approximate NIBSC/WHO concentration, the calculated sample value was multiplied by the concentration ratio.

TABLE 46 Panel 1 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference:NIBSC) IL-1β 86/ 

1.0 IL-2 86/ 

1.1 IL-4

 /656 1.0 IL-6 89/5 

1.0 IL-8 89/520 1.0 IL-10

 /122 1.0 IL-12p70 95/544 1.0 IL-13 94/622 1.0 TNFα

1.0

indicates data missing or illegible when filed

TABLE 47 Panel 2 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference:NIBSC) GM-CSF 88/646 1.08 IL-1α 86/632 1.0 IL-5 90/586 1.0 IL-7 90/530 1.0 IL-15 95/554 0.95 IL-17A 01/420 1.0 TMFβ 87/640 1.0 VEGF 02/285 1.0

TABLE 48 Panel 3 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference:NIBSC) MIP-1α 92/ 

1.0 IL-8 89/ 

1.0 MCP-1 92/ 

0.85

indicates data missing or illegible when filed

TABLE 49 Panel 5 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference:NIBSC) IL-1β

 /668 1.18 IL-2 93/566 0.98 IL-4 91/656 0.89 IL-6

1.0 TNFα

 /532 1.0

indicates data missing or illegible when filed

(a) Normal Sample Testing

Normal mouse serum (rat serum for panel 4), EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were diluted 2- to 4-fold and tested with each panel. Median and range of concentrations for each sample set are displayed below. Concentrations are corrected for sample dilution.

TABLE 50 Panel 1 Sample IL- Type Statistic I 

IL- 

IL-2 IL-4 IL-6 IL-8 IL-10 12p70 IL-13 TNFI Serum Median 3.77 0.0955 0.403 0.00565 0.167 9.61 0.0605 0.0102 0.0994 0.199 (N = 27) (pg/mL) (N Range 1-14 0-14 0-3 0-0 0-27 1-1.121 0-3 0-0 0-3 0-2 (pg/mL) Samples 26 12 15 14 17 27 26 16 12 27 in Quan- titative Range EDTA Median 1.80 0.0538 0.174 0.0166 0.174 0.519 0.167 0.150 0.0 0.735 Plasma (pg/mL) (N = 22) Range 0-23 0-1 0-4 0-0 0-1 0-20 0-3 0-1 0-1 0-2 (pg/mL) Samples 21 22 16 17 15 22 21 17 13 22 in Quan- titative Range Heparin Median 2.87 0.0094 0.0510 0.0 0.114 60.1 0.0798 0.0473 0.0 0.456 Plasma (pg/mL) (N = 27) Range 0-8 0-1 0-3 0-0 0-3 2-2626 0-3 0-0 0-3 0-1 (pg/mL) Samples 27 27 16 13 18 27 23 15 17 27 in Quan- titative Range Citrate Median 3.28 0.0627 0.0097 0.004 

0.190 2.85 0.115 0.0283 0.0 0.481 Plasma (pg/mL) (N = 20) Range 1-15 0-0 0-1 0-0 0-0 0-112 0-2 0-0 0-0 0-3 (pg/mL) Samples 20 11 10 12 10 20 20 11 10 20 in Quan- titative Range Urine Median 0.300 0.350 0.0513 0.0168 0.088 35.185 0.018 0.009 0.0 0.0 (N = 5) (pg/mL) Range 0-1 0-10 0-0 0-0 0-0 1-105 0-0 0-0 0-0 0-0 (pg/mL) Samples 5 5 5 5 5 5 5 5 5 5 in Quan- titative Range ND = Non-detectable

indicates data missing or illegible when filed

TABLE 51 Panel 2 IL- Sample

- 12/IL- Type Statistic CEF IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TNFβ VEGF Serum Median 34.1 1 ND 1 53 1 60 5 ND 9 (N = 20) (pg/mL) Range 11-44  1-62 ND 1-3  13-159 1-3 24-137 5-5 ND  2-187 (pg/mL) Samples in 8 9 0 15 20 20 20 1 0 15 Quantitative Range EDTA Median 1.5 2 1 3 65 2 76 9 ND 95 Plasma (pg/mL) (N = 20) Range 0-1  0-99 1-1 0-40  3-395 1-3  0-973  1-55 ND 18-338 (pg/mL) Samples in 2 18 1 15 17 16 15 5 0 13 Quantitative Range Heparin Median ND 1 45 100 ND 1 5 1 ND 9 Plasma (pg/mL) (N = 20) Range ND 1-1 9-148 35- ND  1-57 1-39 1-2 ND  5-484 (pg/mL) 1109 Samples in 0 2 15 15 0 7 17 19 0 17 Quantitative Range Citrate Median ND ND ND ND ND ND ND ND ND ND Plasma (pg/mL) (N = 20) Range ND ND ND ND ND ND ND ND ND ND (pg/mL) Samples in 0 0 0 0 0 0 0 0 0 0 Quantitative Range Urine Median 2 3 ND 1 27 1 47 5 ND 50 (N =5) (pg/mL) Range 2-2 2-3 ND 1-1  27-27  1-2 47-47  5-5 ND 37-83  (pg/mL) Samples in 1 3 0 2 1 2 1 1 0 4 Quantitative Range

indicates data missing or illegible when filed

TABLE 52 Panel 3 Sample Rotaxin- MIP- Type Statistic Rotaxin

3 TA 

IF-10

IL- 

MC 

M 

M 

Serum Median 41 46 6 28 65 9 547 107 1,246 34 (N = 27) (pg/mL) Range 19-145 7-95 5-9  5-70 27-261 7-202  262-655  76-205 606-3249 11-117 (pg/mL) Samples in 26 27 10 27 27 19 7 27 27 27 Quantitative Range EDTA Median

 17 63 13 79 197 11 769 79 1,309 67 Plasma (pg/mL) (N = 27) Range 37-795  8-153 5-115 13-373 97-676 7-651  340-2478 42-185

 69-2144 12-582 (pg/mL) Samples in 27 27 26 27 27 20 11 27 27 27 Quantitative Range Heparin Median 293 121 31 92 146 41 775 137 1,050 183 Plasma (pg/mL) (N =23) Range  22-1522 10-301 5-147  6-957 91-625 7-2231 234-3281 69-319 589-1963 67-716 (pg/mL) Samples in 27 27 27 27 27 26 7 27 27 27 Quantitative Range Citrate Median 191 51 12 51 77 9 964 135 994 64 Plasma (pg/mL) (N = 20) Range 72-288 19-123 7-19  25-131 36-373 7-30  328-1859 76-242 576-1364 35-161 (pg/mL) Samples in 20 20 19 20 20 9 4 20 20 20 Quantitative Range Urine Median 13 3 ND 1 8 ND 296 80 ND 13 (N = 5) (pg/mL) Range 13-13  2-13 ND 1-1  3-64 ND 296-296  56-122 ND 11-15  (pg/mL) Samples in 1

0 1 3 0 1 5 0 2 Quantitative Range

indicates data missing or illegible when filed

TABLE 53 Panel 5 Sample IL- Type Statistic IFN 

IL-1β IL-2 IL-4 IL-5 IL-6

IL-10 12p70 T 

Serum Median 0.95 2.27 1.02 0.43

21.6 48.3 11.0

 1.0 12.0 (N = 27) (pg/mL) Range  0.34- 1.13- 0.55- 0.23- 0.5 

 - 5.2 

 - 2 

 .1- 5.71- 64. 

 -

 .23- (pg/mL) 2 

3. 

3.9 

1.10 6. 

110. 

101. 

 5. 

97.1 34.4 Samples in 16 16 16 15 16 16 16 16

16 Quantitative Range EDTA Median 41.2 0.86 3.8 

0. 

2. 

 .1 10.5 5 

 .5

 .3 3 

 .5 Plasma (pg/mL) (N = 27) Range 18. 

 - 0.46- 2. 

 - 0.48- 1.5 

 - 1 

 .0- 54.2- 31.5- 50.2- 21.3- (pg/mL) 262.1 2.40 5.8 

0.70 2. 

1 

 4.6

 6. 

74.7 170. 

 7.0 Samples in 15 13 15

15 15 15 15 11 15 Quantitative Range Heparin Median 2 

1.62 4. 

 3 0.75 4.01 115.2 269.4 7 

 .4 85.6 65.3 Plasma (pg/mL) (N =15) Range 156.4- 0.61- 3.35- 0.42- 2.26- 28. 

 - 22 

 .1- 63.7- 38.0- 35.0- (pg/mL) 352.4 2.25 7.36 1.49 5. 

 2 354.6 368.6 104.6 152.0 76.7 Samples in 15 13 15

15 15 15 15 8 15 Quantitative Range Citrate Median 7.04 1.01 3.09 0.73 3. 

4 

6 

30.7 71.2 42. 

Plasma (pg/mL) (N = 20) Range 0. 

  0.45- 0. 

  0.39- 1. 

  6.84-  

 - 5.30- 50.4- 5.45- (pg/mL) 121. 

2.02 5.0 

1.4 

0.24

 4.2

 2.0 68.2 107.4 58.8 Samples in 16 16 15 16 16 16 16 16 15 16 Quantitative Range Urine Median 0.32 0.5 

0.4 

0.43 ND ND 2.31 1.36 101. 

0.63 (N = 10) (pg/mL) Range  0.09- 0.35- 0.4 

 - 0.43- ND ND 1.91- 0.9 

 - 67.3 0.48- (pg/mL) 0.66 1.34 0.65 0.62 2.84 1.53 125.1 3.90 Samples in  

6 3 9 0 0 10 4 9 8 Quantitative Range

indicates data missing or illegible when filed

(b) Stimulated Samples

Panel 1: Freshly collected normal human whole blood was incubated with LPS and simultaneously incubated with peptidoglycan (PG) and Zymosan (ZY) for different time periods and plasma was then isolated. These samples were then tested with panel 1. The dilution adjusted concentrations for each stimulation model is displayed below.

TABLE 54 Panel 1 Incubation IL- Stimulant Time (hr)

IL- 

IL-2 IL-4 IL-6 IL- 

IL-10 12p70 IL-1 

T 

Control 0

0.22 0.68 0.09 0.63

0.37 ND 2.57 Control 6 hr 9.1 4.53 0.31 0.1 1.09

0.45

LPS 3

1028 ND 4.84 8028 5635

2.99 5.14

12 

ND

ND

P 

 A 100 ng 6 hr

14.51 ND ND ND

ND

P 

 A 1 ng 6 hr

ND 0.13

ND

 

indicates data missing or illegible when filed

Panel 2: Freshly collected normal human whole blood was incubated at 37° C. with LPS and PHA for different time periods and plasma was isolated. The samples were tested with panel 2.

TABLE 55 Panel 2 IL- Incubation 12/IL-23 Stimulant Time (hr)

IL- 

IL-5 IL-7 p 

IL-15 IL-16 IL-17A T 

VEG 

Control 0 ND ND ND ND 96 2  73 ND ND ND Control 6 hr ND 12 ND 4 95 2 129 ND ND 44 LPS 3 ND ND ND ND 96 2  73 ND ND ND 12  ND 19 ND 3 6427  2 161 ND 1  3 P 

 A 100 ng 6 hr ND 10 ND 4 75 2 130 ND ND 33 P 

 A 1 ng 6 hr ND 16 ND 4 343  ND 794 ND ND 2096 

indicates data missing or illegible when filed

Panel 3: Freshly collected normal human whole blood was incubated at 37° C. with LPS for different time period and plasma was isolated. The samples were tested with panel 3.

TABLE 56 Panel 3 Incubation Rotarin- M 

 - Stimulant Time (hr) Rotarin M 

 

T 

IP-10 1α IL- 

MCP-1 MDC MCP-4 Control 0 184

11 81    239   10 ND 114 1128 62 LPS (10 3

>4000 5 134 >10 000 >3960 3468 412 1 

89 mg/mL) 12 91 >4000 10 161 >10 000 >3960 2764 250 1210 114 

indicates data missing or illegible when filed

Panel 5: Freshly collected normal pooled mouse whole blood was incubated with LPS and simultaneously incubated with peptidoglycan (PG) and Zymosan (ZY) for different time periods and plasma isolated. Samples were run on panel 5.

TABLE 57 Panel 5 Incubation IL- Stimulant Time (hr)

IL-1β IL-2 IL-4 IL-5 IL-6

IL-10 12p70 T 

None 0 11

3.20 125.00

(Control) LPS 3

151045

12 11.73

3,562.00

None 3

3.49 400.40

31.55

(Control) PG/IY 3 14.05

3.93

None 12 15.01

(Control) PG/IY 12 3076

3.01 2.00

1500.23

indicates data missing or illegible when filed

For panels 1-3, freshly isolated PBMC from normal whole blood was stimulated with LPS, PHA, PWM, Con A, and co-stimulated with CD3 and CD28 antibodies. The samples were then tested with panels 1-3. The dilution adjusted concentrations in pg/mL for each stimulation model is displayed.

TABLE 58 Panel 1 Incubation IL- Stimulant Time (hr) IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 12p70 IL-13 TNF

Control 24 hr 2.55 16.55 0.49 ND ND 4043 0.8 0.53 7.6 1.1

 A 5 mg/mL 24 hr 1382 222 1614 20.28 9052 >1000 136.3 13.05 277.1 355.6 LPS 24 hr 1494 >1000 ND ND 27701 >1000 748 9.8 228.5 >560 10 mg/mL P 

24 hr 5458 5974 995 5.09 29858 205183 1123 3.96 5.5 1276 50 mg/mL a-CD34 + CD28 24 hr 1747 4.11 571.2 11.03 ND 8017 42.9 ND 35 11.35 (545 mg/mL) Co 

  A 24 hr 24558 116.5 3889 11.4 236.9 46528 146 24.04 45.08 263.5 20 mg/mL

indicates data missing or illegible when filed

TABLE 59 Panel 2 IL- Incubation 12/IL- TNF- Stimulant Time (hr)

IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A

VEGF Control 24 hr ND ND ND 7 14 ND 1270 ND ND 1243

 A 5 mg/mL 24 hr

51 291 14 338 ND 960 1677 9 1147 LPS 24 hr 17 940 2 11 343 ND 794 ND ND 2096 10 g/mL P 

  24 hr 66 811 31 9 526 ND 782 658 2 2194 50 mg/mL a-CD-34 + CD28 24 hr 78 339 10 132 ND 1046 321 27 804 (545 mg/mL) Co 

  A 24 hr 212 272 194 11 1390 ND 1252 816 53 1299 20 mg/mL

indicates data missing or illegible when filed

TABLE 60 Panel 3 Incubation Rotaxin- Time (hr) Rotaxin MEP-1β

T 

IP- 

 EP-1α IL- 

MCP-1 MDC MCP-4 Control  6 hr 140 131 33 132 312 41 ND 80 1157 47 Control 24 hr 127 75 31 159 2322 34  3742 141 769 59

 A (100  6 hr 197 1753 31 147 372 117 NaN 71 942 55 mg/mL)

 A (1  6 hr 149 >4100 21 141 1653 1853 NaN 443 940 55 mg/mL)

 A (5 24 hr 130 19163 NaN 683 107930 10374 192588 80059 2986 278 mg/mL) LPS (10 24 hr 84 59243 NaN 258 3036 48161 182842 355 315 149 mg/mL)

 (50 24 hr 131 59467 NaN 232 4169

195681 465 360 123 mg/mL) CD3 + CD28 24 h 143 1358 NaN 1250 75234 534  4646 6844 2543 507 (5 mg/mL each) Co 

  A (20 24 hr 230 4347 NaN 1307 105145 700  35480 43377 3889 320 mg/mL)

indicates data missing or illegible when filed

For panels 1-3, human acute monocyte leukemia cell line (THP-1 cell line) was stimulated with LPS for six and 16 hours. The supernates were then isolated and tested with panels 1-3. The dilution adjusted concentrations in pg/mL for each sample is displayed below.

TABLE 61 Panel 1 Incubation IL- Stimulant Time (hr) I 

IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 12p70 IL-15 TN 

Control  0 hr 1.09 20.48 0.345 0.07 0.19 449.8 0.44  0.38 1.41 9.91 LPS  6 hr 1.1  645.5 11.5 ND ND 61066 97.2 11.12 0.87 12472 16 hr 0.67 423   ND ND ND 69638 15.5 ND 1.06 2915

indicates data missing or illegible when filed

TABLE 62 Panel 2 IL- Incubation 12/IL- Stimulant Time (hr) GM-C 

IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TNF

VEGF Control  0 hr ND ND ND ND ND ND 205 ND ND 1995 LPS  6 hr ND 47 ND ND 55 ND 421 ND ND 276 16 hr ND 22 ND ND 234 ND 552 ND ND >1070

indicates data missing or illegible when filed

TABLE 63 Panel 3 Incubation Rotaxin- Stimulant Time (hr) Rotaxin MIP-1β 1 T 

IP-10 MCP-1α IL- 

MCP-1

MCP-4 Control  0 hr 26 993 12 ND 95 70   1132 205   148 ND LPS  6 hr ND >4000 ND ND 324 >3960 45 546 577

 57 16 hr ND >4000 ND 20 687 2759 52 262 1290  >40 000 332

indicates data missing or illegible when filed

For panel 5, a mouse monocyte macrophage cell line (J774A.1) and a mouse leukemic monocyte macrophage cell line (RAW 264.7) were stimulated with different stimulants. The J774A.1 cell line stimulation was for four house while the RAW cell line stimulation was for six hours. The lysates were collected and run on panel 5. The concentrations are listed in pg/mL and normalized for 50 ug of lysate per well.

TABLE 64 Panel 5 IL- Cell Line Stimulant IFNγ IL-1β IL-2 IL-4 IL-5 IL-6

IL-10 12p70 T 

  J774A.1 None ND 1.9 ND ND ND

ND  34 ND 812 J774A.1 5 μg/mL LPS ND

3.9 ND ND 62 529 107 320 ND >10 000 J774A.1 5 μg/mL 

ND 10 674 2.8 ND ND

111 209 ND >10 000 J774A.1  1 ng/mL LPS ND

ND ND ND

ND 364 J774A.1 100 ng/mL  

ND

ND ND ND

ND

ND 264 RAN 264.7 100 ng/mL LPS ND

ND

ND >10 000

indicates data missing or illegible when filed

The following calibrator blends were used in each panel as follows:

TABLE 65 Panel 1 Calibrator Sequence Expression System IFNγ Gln 

-Gln166 E. coli IL-1β Ala 

-Ser269 E. coli IL-2 Ala21-Thr153 E. coli IL-4

 -Ser153 E. coli IL-6 Pro29-Met212 E. coli IL-8

 -Ser99 E. coli IL-10 Ser19-

5f21 insect cells IL-12p70 IL-12p 

5f21 insect cells IL-12p35 

IL-13 Gly21-Asn132 E. coli TNFα

 -Leu233 E. coli

indicates data missing or illegible when filed

TABLE 66 Panel 2 Calibrator Sequence Expression System GM-CSF ala18-Glu144 E. coli IL-1α Ser113-Ala 

E. coli IL-5 Ile20-Ser134 5f21 insect cells IL-7 Asp26-His 

E. coli IL-12/IL-23 p40 Ile23-Ser 

5f21 insect cells IL-15

 -Ser162 E. coli IL-16 Pro2-Ser 

E. coli IL-17A

 -Ala155 E. coli TNFβ

 -Leu205 E. coli VEGF Ala21-

5f21 insect cells

indicates data missing or illegible when filed

TABLE 67 Panel 3 Calibrator Sequence Expression System Eotaxin Gly24-Pro 

E. coli MIP-1β Ala24-Asn 

E. coli Eotaxin-3 Thr24-Leu 

E. coli TARC Ala24-Ser 

E. coli IP-10 Val22-Pro 

E. coli MIP-1α Ala 

E. coli IL-8

-Ser99 E. coli MCP-1 Gln 

-Thr99 E. coli

Gly25-Gln 

E. coli MCP-4 Gln24-Thr 

E. coli

indicates data missing or illegible when filed

TABLE 68 Panel 4 Calibrator Sequence Expression System IFNγ Gl 

E. coli IL-2 Ala21-Gln155 E. coli IL-4

E. coli IL-1β

E. coli IL-5

E. coli IL-6

-Thr211 E. coli

/GRO Ala25- 

E. coli IL-10 Ser19- 

E. coli IL-11 Thr19- 

E. coli TNFα

-Leu235 E. coli

indicates data missing or illegible when filed

TABLE 69 Panel 5 Calibrator Sequence Expression System IFNγ

 -Cys155 E. coli IL-1β

E. coli IL-2 Ala21- 

E. coli IL-4

E. coli IL-5

 -Gly133 5f21 insect cells IL-6

 -Thr211 E. coli KC/GRO

 -Lys96 E. coli IL-10 Ser19- 

E. coli IL-12p70 Met23-Ser 335 

5f21 insect cells Arq23-Ala215 

TNFα

 Leu235 E. coli

indicates data missing or illegible when filed

The following antibodies, capture and detection, were used in each panel as follows:

TABLE 70 Panel 1 Source Species MSD Detection Analyte MSD Capture Antibody Antibody IFNγ Mouse Monoclonal Mouse Monoclonal IL-1β Mouse Monoclonal Goat Polyclonal IL-2 Mouse Monoclonal Mouse Monoclonal IL-4 Mouse Monoclonal Mouse Monoclonal IL-6 Mouse Monoclonal Goat Polyclonal IL-8 Mouse Monoclonal Goat Polyclonal IL-10 Mouse Monoclonal Mouse Monoclonal IL-12p70 Mouse Monoclonal Mouse Monoclonal IL-13 Rat Monoclonal Mouse Monoclonal TNFα Mouse Monoclonal Goat Polyclonal

TABLE 71 Panel 2 Source Species MSD Detection Analyte MSD Capture Antibody Antibody GM-CSF Mouse Monoclonal Rat Monoclonal IL-1α Mouse Monoclonal Goat Polyclonal IL-5 Mouse Monoclonal Mouse Monoclonal IL-7 Mouse Monoclonal Goat Polyclonal IL-12/IL-23 p40 Mouse Monoclonal Mouse Monoclonal IL-15 Mouse Monoclonal Mouse Monoclonal IL-16 Mouse Monoclonal Goat Polyclonal IL-17A Mouse Monoclonal Goat Polyclonal TMFβ Mouse Monoclonal Mouse Monoclonal VEGF Mouse Monoclonal Mouse Monoclonal

TABLE 72 Panel 3 Source Species MSD Detection Analyte MSD Capture Antibody Antibody Eotaxin Mouse Monoclonal Mouse Monoclonal MIP-1β Mouse Monoclonal Mouse Monoclonal Eotaxin-3 Mouse Monoclonal Mouse Monoclonal TARC Mouse Monoclonal Mouse Monoclonal IP-10 Mouse Monoclonal Mouse Monoclonal MIP-1α Mouse Monoclonal Mouse Monoclonal IL-8 Mouse Monoclonal Goat Polyclonal MCP-1 Mouse Monoclonal Mouse Monoclonal MDC Mouse Monoclonal Mouse Monoclonal MCP-4 Mouse Monoclonal Mouse Monoclonal

TABLE 73 Panel 4 Source Species MSD Detection Analyte MSD Capture Antibody Antibody IFNγ Mouse Monoclonal Goat Polyclonal IL-2 Mouse Monoclonal Goat Polyclonal IL-4 Mouse Monoclonal Goat Polyclonal IL-1β Mouse Monoclonal Goat Polyclonal IL-5 Rat Monoclonal Rat Monoclonal IL-6 Mouse Monoclonal Goat Polyclonal KC/GRO Mouse Monoclonal Goat Polyclonal IL-10 Mouse Monoclonal Goat Polyclonal IL-15 Mouse Monoclonal Goat Polyclonal TNFα Hamster Monoclonal Goat Polyclonal

TABLE 74 Panel 5 Source Species MSD Detection Analyte MSD Capture Antibody Antibody IFNγ Rat Monoclonal Rat Monoclonal IL-1β Mouse Monoclonal Goat Polyclonal IL-2 Rat Monoclonal Rat Monoclonal IL-4 Rat Monoclonal Rat Monoclonal IL-5 Rat Monoclonal Rat Monoclonal IL-6 Rat Monoclonal Goat Polyclonal KC/GRO Rat Monoclonal Goat Polyclonal IL-10 Rat Monoclonal Goat Polyclonal IL-12p70 Rat Monoclonal Rat Monoclonal TNFα Hamster Monoclonal Goat Polyclonal

Various publications and test methods are cited herein, the disclosures of which are incorporated herein by reference in their entireties, In cases where the present specification and a document incorporated by reference and/or referred to herein include conflicting disclosure, and/or inconsistent use of terminology, and/or the incorporated/referenced documents use or define terms differently than they are used or defined in the present specification, the present specification shall control.

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1. A kit for the analysis of a cytokine panel comprising (a) a multi-well assay plate selected from: (i) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the human analytes are bound: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFalpha; (ii) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A; (iii) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, MCP-4; (iv) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following rat analytes are bound: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha; or (v) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following mouse analytes are bound: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.
 2. The kit of claim 1 wherein said kit further comprises one or more diluents.
 3. The kit of claim 1 wherein said detection antibodies are labeled with an electrochemiluminescent (ECL) label.
 4. The kit of claim 3 wherein said kit further comprises an ECL read buffer.
 5. The kit of claim 3 wherein said discrete binding domains are positioned on an electrode within said well.
 6. The kit of claim 1 wherein said set of calibrator proteins comprise a lyophilized blend of proteins.
 7. The kit of claim 1 wherein said set of calibrator proteins comprise a liquid formulation of calibrator proteins.
 8. A 10-spot 96-well multi-well plate, wherein each plate comprises a plate top, a plate bottom, and each well comprises a spot pattern, wherein the plate meets the following specifications: (a) a length range of 3.8904-3.9004 inches; (b) a width range of 2.4736-2.4836 inches; and (c) well to well spacing of 0.3513-0.3573 inches.
 9. A lot of plates of claim
 8. 10. A kit comprising a plate of claim 8, wherein said spot pattern comprises ten discrete binding domains to which capture antibodies to one of the following sets of analytes are bound: (a) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFalpha; (b) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A; (c) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, MCP-4; (d) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha; (e) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha; said kit further comprising (i) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (ii) in one or more vials, containers, or compartments, a set of calibrator proteins.
 11. A plate of claim 8, wherein said plate exceeds said specifications.
 12. A plate of claim 8, wherein (a) and (b) are measured from a center of a spot pattern in a first well, A1, to a center of a spot pattern of an outermost well, A12, of said plate.
 13. A 10-spot 96-well multi-well plate, wherein each plate comprises a plate top, a plate bottom, an x- and y-axis of the plate top and bottom, and each well comprises a spot pattern, wherein the plate meets the following specifications: Δx≦0.2 mm, Δy≦0.2 mm, and α≦0.1°, wherein (a) Δx is the difference between a center of the spot pattern and a center of a well along the x axis of the plate; (b) Δy is the difference between the center of a spot pattern and a center of the well along the y axis of the plate; and (c) α is a counter-clockwise angle between the x axis of the plate bottom and the x axis of the plate top.
 14. A lot of plates of claim
 13. 15. A kit comprising a plate of claim 13, wherein said spot pattern comprises ten discrete binding domains to which capture antibodies to one of the following sets of analytes are bound: (a) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFalpha; (b) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A; (c) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, MCP-4; (d) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha; (e) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha; said kit further comprising (i) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (ii) in one or more vials, containers, or compartments, a set of calibrator proteins.
 16. A plate of claim 13, wherein said plate exceeds said specifications.
 17. A kit for the analysis of two or more cytokine panels comprising: (a) two or more multi-well assay plates each comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to a set of analytes are bound, wherein said set of analytes is selected from the group consisting of: (i) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha; (ii) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A; (iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4; (iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or (v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.
 18. The kit of claim 17 wherein said detection antibodies are labeled with an electrochemiluminescent (ECL) label.
 19. The kit of claim 18 wherein said discrete binding domains are positioned on an electrode within said well.
 20. The kit of claim 18 wherein said capture antibodies and/or detection antibodies have been subjected to an analytical testing method selected from the group consisting of CIEF, SEC-MALS, DLS, denaturing/non-denaturing gels, and Experion.
 21. A method of manufacturing a lot of kits used in the analysis of a cytokine panel, wherein said kit comprises qualified detection and capture antibodies specific for one of the following sets of analytes: (i) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha; (ii) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A; (iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4; (iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or (v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha; said method comprising the steps of subjecting a subset of kits in said lot to plate coating uniformity testing and passing said lot based on results of said uniformity testing.
 22. The method of claim 21 wherein said lot meets a specification selected from the group consisting of: (a) average intraplate CV of ≦10%; (b) maximum intraplate CV of ≦13%; (c) average uniformity metric of ≦25%; (d) maximum uniformity metric of ≦37%; (e) CV of intraplate averages of ≦18%; (f) lower signal boundary of >1500; and (g) upper signal boundary of <10⁶.
 23. The method of claim 21 wherein said lot meets the following specifications: (a) average intraplate CV of ≦10%; (b) maximum intraplate CV of ≦13%; (c) average uniformity metric of ≦25%; (d) maximum uniformity metric of ≦37%; (e) CV of intraplate averages of ≦18%; (f) lower signal boundary of >1500; and (g) upper signal boundary of <10⁶.
 24. A method of manufacturing a kit used in the analysis of a cytokine panel, wherein said kit comprises qualified detection and capture antibodies specific for one of the following sets of analytes: (i) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha; (ii) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A; (iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4; (iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or (v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha; said method comprising the steps of: (a) subjecting a preliminary set of detection antibodies specific for said mouse analytes to CIEF, DLS, and Experion; (b) selecting qualified detection antibodies from said preliminary set of detection antibodies based on said CIEF, DLS, and Experion testing; (c) subjecting a preliminary set of capture antibodies specific for said mouse analytes to CIEF, DLS, and Experion; and (b) selecting qualified capture antibodies from said preliminary set of capture antibodies based on said CIEF, DLS, and Experion testing.
 25. The method of claim 24, wherein said method further comprises subjecting said preliminary set of detection antibodies to an additional analytical method selected from the group consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof.
 26. The method of claim 24, wherein said method further comprises subjecting said preliminary set of detection antibodies to an additional analytical method consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, and SEC-MALS.
 27. The method of claim 24, wherein said method further comprises subjecting said preliminary set of capture antibodies to an additional analytical method selected from the group consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof.
 28. The method of claim 24, wherein said method further comprises subjecting said preliminary set of capture antibodies to an additional analytical method consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, and SEC-MALS.
 29. The method of claim 24 wherein said method further comprises subjecting each of said preliminary set of detection and capture antibodies to an additional analytical method consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, and SEC-MALS. 